Process for identifying substances which modulate the activity of hyperpolarization-activated cation channels

Abstract

The present invention provides a process for identifying substances that modulate the activity of hyperpolarization-activated cation channels, and the use of this process.

Parent Case Text

This is a continuation of application Ser. No. 09/779,587, filed Feb. 9,2001, now abandoned, which is incorporated herein by reference.

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of biological cell-to-cell communication and electrochemical signalling between biological cells. In particular, the present invention provides a process for identifying substances that modulate the activity of hyperpolarization-activated cation channels, and the use of this process.

2. Description of the Relevant Art

Some genes of murine and human hyperpolarization-activated cation channels are already known. Examples include muHCN2(muHAC1) (Ludwig et al. (1998)), huHCN4 (Ludwig et al. (1999)), huHCN2 (Vaccari, T. et al. (1999) Biochim. Biophys. Acta 1446(3): 419-425), and those disclosed in WO 99/32615 and WO 99/42574. See, also, Tables 1-6 herein.

Ludwig et al. (1998) have shown that muHCN2 can be transfected transiently in HEK293 cells, and that the corresponding channel in the transfected cells can be examined easily by electrophysiological methods (patch-clamp studies). The electrophysiological properties of the cloned channel correspond to the l.sub.f or l.sub.h current described in pacemaker cells, which had hitherto not been known on a molecular level (Ludwig et al. (1998), Biel et al. (1999)). The channel activates when the holding potential is changed toward hyperpolarization (potential at about B100 to B160 mV). However, the patch-clamp technique cannot be automated and is not suitable for high-throughput screening (HTS).

Using suitable dyes, ion currents can be measured in an FLIPR (fluorescence imaging plate reader; Molecular Devices, Sunnyvale Calif., USA). Influx or efflux of ions leads to changes in the membrane potential, which can be measured in high-throughput screening in an FLIPR using suitable fluorescent dyes. However, in contrast to the patch-clamp method, it is not possible to generate voltage changes in the FLIPR. Voltage changes are, however, an essential prerequisite for the activation of hyperpolarization-activated cation channels.

SUMMARY OF THE INVENTION

For the examination of the largest possible number of substances, we have developed a process that permits, among other things, high-throughput screening (HTS) for modulators of a hyperpolarization-activated cation channel.

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations used herein are listed in Table 7 below.

The present invention provides a way to hyperpolarize cells that express a hyperpolarization-activated cation channel (i.e. to activate the hyperpolarization-activated cation channel) and to maintain this hyperpolarization of the cell, for example, until a measurement of membrane potential can be taken. Under physiological conditions, a hyperpolarization of the cell that is sufficient to activate a hyperpolarization-activated cation channel is reversed by the activity of that channel. Only when hyperpolarization can be maintained is it possible to measure, for example in an FLIPR, the depolarization of the cell caused under suitable conditions by a substance that modulates the activity of the hyperpolarization-activated cation channel.

Generally speaking, the present invention provides a process for examining hyperpolarization-activated cation channels. In the process, cells that express the hyperpolarization-activated cation channels are hyperpolarized (i.e. the hyperpolarization-activated cation channel is activated) and this hyperpolarization of the cells, which is reversed under physiological conditions by the activity of the hyperpolarization-activated cation channel, is maintained. By exclusion of extracellular sodium ions, the activated channel is unable to transport sodium ions into the cells, i.e. to depolarize the cells. If, simultaneously or even prior to the addition of the sodium ions, substances are added that modulate the activity of the hyperpolarization-activated cation channel, the depolarization is affected. For example, compared to when only sodium ions are added, depolarization is increased in the case of HCN activators (for example forskolin) and reduced in the case of HCN inhibitors (for example zatebradine=3-[3-[[2-(3 ,4-dimethoxyphenyl)ethyl]methylamino]propyl]-1,3,4,5-tetrahydro-7,8-dimeth oxy-2H-3-benzazepin-2-one; Reiffen et al. (1990)).

By measuring the depolarization of the cells or the changes of their membrane potential, it is possible to identify substances that modulate the activity of the hyperpolarization-activated cation channel.

In one aspect, the invention generally provides a process for identifying substances that modulate the activity of hyperpolarization-activated cation channels, wherein a) cells which express a hyperpolarization-activated cation channel are used; b) the cells are hyperpolarized in the presence of a potential-sensitive fluorescent dye using an isoosmolar sodium-ion-free buffer; and c) the change in the membrane potential of the cells following simultaneous addition of sodium ions and the substance to be examined is detected and recorded.

Thus, in embodiments, the invention provides a process for identifying substances that modulate the activity of hyperpolarization-activated cation channels, wherein the process comprises a) providing, in a suitable container, cells that express a hyperpolarization-activated cation channel; b) hyperpolarizing the cells in the presence of a potential-sensitive fluorescent dye and an isoosmolar sodium-ion-free buffer; c) optionally, determining the membrane potential of the cells; d) simultaneously adding sodium ions and a sample containing at least one substance to be tested for its ability to modulate the activity of the cation channel; e) determining the membrane potential of the cells; f) determining whether the membrane potential changed upon simultaneous addition of sodium ions and the substance(s); and g) optionally, recording the change in membrane potential,

wherein a change in membrane potential indicates the presence of at least one substance in the sample that modulates the activity of the cation channel.

A suitable container is any container, vessel, receptacle, etc. that can be used to hold the reagents and samples to be used in the assay. Suitable containers are disclosed in, or identifiable from, literature provided by manufacturers of equipment designed to determine membrane potentials. Such equipment is publicly available and well known to those of skill in the art.

In embodiments where step "c)" is not performed, a parallel assay, using the same strain of cells at the same concentration in the same assay composition, can be run to determine the membrane potential of the cells in the absence of the sample suspected of containing at least one substance that can modulate the activity of a cation channel.

In embodiments, the assay is a high-throughput assay.

In another aspect, the invention generally provides a process for identifying substances that modulate the activity of hyperpolarization-activated cation channels, wherein a) cells which express a hyperpolarization-activated cation channel are used; b) the cells are hyperpolarized in the presence of a potential-sensitive fluorescent dye using an isoosmolar sodium-ion-free buffer; c) the cells are incubated with a substance to be examined; and d) the change in the membrane potential of the cells after addition of sodium ions is detected and recorded.

Thus, in embodiments, the invention provides a process for identifying substances that modulate the activity of hyperpolarization-activated cation channels, wherein the process comprises a) providing, in a suitable container, cells that express a hyperpolarization-activated cation channel; b) hyperpolarizing the cells in the presence of a potential-sensitive fluorescent dye and an isoosmolar sodium-ion-free buffer; c) optionally, determining the membrane potential of the cells; d) incubating the cells with a sample containing at least one substance to be tested for its ability to modulate the activity of the cation channel; e) optionally, determining the membrane potential of the cells; f) optionally, determining whether the membrane potential changed upon addition of the substance(s) to be tested; g) adding sodium ions; h) determining the membrane potential of the cells; i) determining whether the membrane potential changed upon addition of the sodium ions; and j) optionally, recording the change in membrane potential, wherein a change in membrane potential between the time before the sodium ions are added and after the sodium ions are added indicates the presence of at least one substance in the sample that modulates the activity of the cation channel.

Extracellular potassium ions can be included in the assay. In certain situations, these ions can improve the function of the hyperpolarization-activation cation channels. For example, they might be included when HCN (HAC) channels are being used in the process. Thus, in embodiments of the present invention, the isoosmolar sodium-ion-free buffer comprises potassium ions (K.sup.+). In embodiments, the buffer comprises potassium ions in the form of potassium chloride. In embodiments, the buffer comprises potassium ions at a concentration of at least about 0.5 mM K.sup.+. In embodiments, the buffer comprises potassium ions at a concentration of at least about 0.8 mM K.sup.+. In embodiments, the buffer comprises potassium ions at a concentration of about 2 mM. In embodiments, the buffer comprises potassium ions at a concentration of about 5 mM.

In embodiments, the isoosmolar sodium-ion-free buffer comprises at least one cation that is not able to cross the membrane in amounts that correspond to the normal extracellular sodium ion concentration. For example, the buffer can comprise choline, for example in the form of choline chloride, or NMDG (N-methyl-D-glucamine). In embodiments, the isoosmolar sodium-ion-free buffer comprises both potassium ions and at least one cation that is not able to cross the membrane in amounts that correspond to the normal extracellular sodium ion concentration.

In embodiments, the isoosmolar sodium-ion-free buffer comprises a potential-sensitive dye, for example a potential-sensitive fluorescent dye. Included among these are oxonol derivatives, such as 3-bis-barbituric acid oxonol. Thus, in embodiments, the isoosmolar sodium-ion-free buffer comprises potassium ions, at least one cation that is not able to cross the membrane in amounts that correspond to the normal extracellular sodium ion concentration, and a potential-sensitive dye.

In embodiments, the buffer comprises potential-sensitive fluorescent dyes that are suitable for examining the membrane potential of nonexcitable cells. Examples of such dyes include, but are not limited to, potential-sensitive slow-response dyes. Non-limiting examples of such potential-sensitive slow-response dyes include bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBac.sub.4 (3)], bis-(1,3-diethylthiobarbituric acid)trimethine oxonol [DiSBac.sub.2 (3)] or bis-(1,3-dibutylbarbituric acid)pentamethine oxonol [DiBac.sub.4 (5)]. Other known and suitable potential-sensitive dyes include, but are not limited to, fast-response dyes (for example, of the styrylpyridinium type), which are used in certain embodiments in conjunction with excitable cells, such as neurons, cardiac cells, etc. These potential-sensitive dyes react in the millisecond range and are not particularly sensitive (2-10% fluorescence change per 100 mV potential change). Other suitable dyes include slow-response dyes of the carbocyanine type. Non-limiting examples of these slow-response dyes include diOC5(3)-3,3'-dipentyloxacarbocyanine iodide, diOC6(3)-3,3'-dihexyloxacarbocyanine iodide, etc.), JC-1 (5,5',6,6'-tetrachloro-1,1'-3,3'-tetraethylbenzimidazolecarbocyanine iodide), and rhodamine 123. In embodiments, these slow-response potential-sensitive dyes are used in studies of the membrane potential of mitochondria.

One embodiment of the invention relates to the use of the fluorescent dye from the FLIPR Membrane Potential Assay Kit (Molecular Devices, Sunnyvale, Calif., USA). The fluorescence of this dye can be measured using a standard emission filter, which is transparent between about 510 and about 580 nm. In embodiments, fluorescence of this dye is measured using a filter that is transparent above about 550 nm. The manufacturer of this dye and kit disclose a number of advantages of their product, over, for example, assays based on DiBac.sub.4 (3), and these advantages can be applicable to the present invention.

Some of these advantages include: 1) the measurement of membrane potentials with the kit is not temperature sensitive, in contrast to DiBac.sub.4 (3), where the temperature has to be equilibrated prior to the actual measurement in the FLIPR; 2) the volume added in the FLIPR can be greater than that in the case of DiBac.sub.4 (3), where usually all substances have to be added in a 10-fold concentrated form; 3) the measurements can be carried out much more rapidly, since the kit requires a much shorter time to reach the steady state than DiBac.sub.4 (3), which usually requires between 10 and 30 minutes; 4) for many measurement protocols, a washing step prior to the addition of the dye is no longer required; and 5) the dye does not have to be present in each solution.

In embodiments of the present invention, the first two advantages are relied upon because these two advantages can be applied to assays of hyperpolarization-activated cation channels. The first two advantages can also be applied to embodiments of the invention that are directed to high-throughput screening, since screening of a large number of samples at once can be complicated and/or time consuming. For example, in embodiments where FLIPR II, which allows the measurement in 384-well plates and which is preferably employed for high-throughput screening thermostating, is used, these first two advantages can reduce the complications and time necessary to perform the assay process. In the case of poorly soluble substances, it is furthermore an advantage if they can be added to the cells in five-, three-, or even two-fold concentrated form instead of 10-fold concentrated form, as is typical with DiBac.sub.4 (3).

In the processes of the present invention, cells having an elevated intracellular cAMP concentration can be used. Elevated intracellular cAMP concentrations can be achieved, for example, by adding cAMP derivatives that are able to cross the membrane. Non-limiting examples of such derivatives include dibutyryl-cAMP and 8-bromo-cAMP. As a further non-limiting example, the intracellular cAMP concentration can be increased by the addition of an adenylate cyclase activator, for example forskolin. When forskolin is used, successful results can be obtained when it is supplied in concentrations of less than about 100 .mu.M. For example, forskolin can be used at a concentration of between about 1 .mu.M and about 100 .mu.M. It can also be used at concentrations less than about 1 .mu.M. In embodiments, it is used at a concentration of about 10 .mu.M. In principle, it is also possible to use all substances or ligands that activate adenylate cyclase by signal transduction in the cell line employed (for example ligands for b-adrenergic receptors, such as adrenalin, isoproterenol, noradrenalin, etc., if the cell has endogenous b-adrenergic receptors).

To depolarize the membrane potential, Na.sup.+ (which can be supplied in the form of NaCl, for example) is added in the FLIPR to the cells which have hyperpolarized by the sodium-ion-free buffer. In embodiments, the Na.sup.+ is added to achieve a final Na.sup.+ concentration of about 20-100 mM. In embodiments, it is added to achieve a final Na.sup.+ concentration of about 50 mM. In embodiments where the FLIPR Membrane Potential Assay Kit (Molecular Devices, Sunnyvale, Calif., USA) is used, the final Na.sup.+ concentration can be about 20-100 mM. For example, it can be about 40-80 mM.

In embodiments, the invention relates to processes in which the hyperpolarization-activatable cation channel is an HCN1, HCN2, HCN3, HCN4 channel (where HAC1=HCN2, HAC2=HCN1, HAC3=HCN3 and HAC4=HCN4) or a KAT1 (=AKT) channel (hyperpolarization-activated potassium channel from Arabidopsis thaliana); a heteromultimer of these channels (i.e. a channel which is composed of subunits of different hyperpolarization-activated cation channels); or a chimeric hyperpolarization-activated cation channel (i.e. a synthetic channel in which individual subunits are composed of parts of different channels or hyperpolarization-activated cation channels). The hyperpolarization-activated cation channel is preferably a human hyperpolarization-activated cation channel, for example huHCN2, (SEQ ID NO. 1, SEQ ID NO. 2) or huHCN4 (SEQ ID NO. 3, SEQ ID NO. 4), or a murine hyperpolarization-activated cation channel muHCN2 (SEQ ID NO. 5, SEQ ID NO. 6). See Tables 1-6. On the amino acid level, the identity between muHCN2 and huHCN2 is 94.8%. In principle, the process is suitable for all cation channels which are activated by hyperpolarization. For example, it is suitable for HCN1-4 (or HAC1-4; see Biel et al. (1999)).

The cells can be any eukaryotic cells. For example, the cells can be mammalian cells, such as CHO or HEK293 cells. In embodiments, CHO cells or another cell line having comparably few endogenous potassium channels are used, since endogenous potassium channels might interfere with the measurement, for example, in the FLIPR. In other embodiments cells whose endogenous potassium channels are not functionally expressed (for example the corresponding knock-out cells) are used.

The cells can, but do not necessarily, contain nucleic acids (i.e., RNA, DNA, PNA) that code for the hyperpolarization-activated cation channel. In embodiments, the cells contain DNA. In embodiments, the cells contain RNA. In embodiments, the cells contain a eDNA of a hyperpolarization-activated cation channel in a suitable plasmid. Such cells can be prepared by transfecting the original cell line with a plasmid that contains the cDNA of a hyperpolarization-activated cation channel. Other techniques can be used as well. Techniques for introducing heterologous nucleic acids into cells are well known and widely practiced by those of skill in the art, and thus need not be detailed here.

In the case of the hyperpolarization-activated cation channels, it is an object of the invention to detect, and optionally, record changes in the membrane potential of the cells, where the changes are the result of the activation or the inhibition of these channels. Detection can utilize bis-barbituric acid oxonols. Three bis-barbituric acid oxonols (see, for example, "Handbook of Fluorescent Probes and Research Chemicals", 6th edition, Molecular Probes, Eugene Oreg., USA), which are mainly referred to as DiBac dyes, form a family of potential-sensitive dyes having excitation maxima at 490 nm (DiBac.sub.4 (3)), 530 nm (DiSBac.sub.2 (3)), and 590 nm (DiBac.sub.4 (5)). The dyes get into depolarized cells by binding to intracellular proteins or membranes, leading to increased fluorescence and a red shift. Hyperpolarization results in the expulsion of the anionic dyes and thus in a decrease in fluorescence. This decrease in fluorescence can be measured, for example, with the measuring device FLIPR. Accordingly, one embodiment of the invention relates to the measurement of the membrane potential in a Fluorescent Imaging Plate Reader (FLIPR).

The FLIPR (for: Fluorescent Imaging Plate Reader; Manufacturer: Molecular Devices, Sunnyvale, Calif., USA) is a measuring device that allows the simultaneous measurement of changes of the fluorescence intensity in all wells of a microtiter plate. The dyes used are excited at about 488 nm using an argon laser, which is integrated into the system. The standard emission filter of the system is transparent in the range from 510 B 580 nm. The emitted fluorescence is registered using a CCD camera, and the system permits the simultaneous recording, within an interval of about one second, of the fluorescence in all wells of a 96-well or 384-well microtiter plate. Using a built-in pipettor, it is even possible to determine the fluorescence during the addition of the substance, which can be beneficial, for example, in the case of rapid processes. By means of special optics, the fluorescence can be registered in a layer of only about 50 mm, but not in the entire well. This can be beneficial for background reduction in all measurements where the fluorescent dye is also present extracellularly. Such a situation can exist, for example, in the measurement of changes in membrane potential using DiBac dyes. Standard applications of the system are measurements of the intracellular calcium concentration or the membrane potential of cells. Among the dyes mentioned above, DiBac.sub.4 (3), which, owing to its excitation maximum, is most suitable for the argon laser in the FLIPR, has the highest sensitivity for voltage differences.

Since the DiBac.sub.4 (3) takes some time to come to equilibrium, the measurement can be taken after a certain incubation time. In embodiments, the incubation temperature is at or about the optimal temperature for growth and metabolism of the biological cells being used in the assay. For example, the incubation temperature can be at or about 37.degree. C. Incubation time can be varied to achieve complete or uniform sample temperature. In embodiments, the sample can be incubated for at least about 10 minutes. In embodiments, the sample is incubated for about or precisely 30 minutes.

Although results can be obtained at any time desired, in order to obtain as reliable of a result as possible or practical, the results should be determined and, optionally, recorded as quickly as possible after each incubation step. This is because cooling of the dye solution might affect the result of the measurement. Thus, prior to any measurement, the composition to be measured can be incubated at a chosen temperature for a period of time that is sufficient to equilibrate the temperature of the composition at a desired level. For example, the composition can be incubated for at least about one minute, or at least about two, three, for, five, or even more minutes. Included are incubation periods prior to initial measurements (e.g., to determine base-line levels of activity or membrane potential). As with the other incubation periods, this pre-incubation phase can be carried out to compensate for temperature variations on the microtiter plate.

In embodiments where FLIPR is used, the measurement is typically carried out using the temperature parameters preset by the FLIPR manufacturer for the measurement of membrane potentials (about 37.degree. C.). However, this is a guideline, and those practicing the invention can alter the temperature to achieve maximal results. Such temperature modifications are well within the skill of those in the art, and do not represent undue experimentation. In embodiments, the parameters preset by the FLIPR manufacturer are followed essentially precisely.

Although variations in volume can be accounted for, in the FLIPR, in embodiments of the present invention, the volume of the reaction solution in which the process is carried out is changed as little as possible. In embodiments where DiBac.sub.4 (3) is used, the DiBac.sub.4 (3) signal is most reproducible if only relatively small volume changes take place in the FLIPR; thus, the volume is typically maintained throughout, to the extent possible and practicable. Accordingly, in these embodiments, the substances to be tested are added as concentrated solutions. In embodiments, they are added at a concentration of at least about 2-fold. For example, they can be added in about a five-fold, ten-fold, or even greater concentrated form to the DiBac.sub.4 (3)-dyed cells.

Since the fluorescence measurement with the FLIPR Membrane Potential Assay Kit is not temperature-sensitive, it can be carried out simply at room temperature. This can be advantageous, for example, in embodiments that utilize the FLIPR II, which allows measurements with 384-well microtiter plates.

In embodiments, the HCN channels are activated by hyperpolarization (for example HCN2 at B100 mV to about 50%) and cause a depolarization of the cells. By increasing the intracellular cAMP concentration (for example with dibutyryl-cAMP or with forskolin), the value of the half-maximal activation can be shifted by about 10 mV to more positive potentials (Ludwig et al., 1998).

Electrophysiologically, HON channels can be studied easily on stably transfected cells using the patch-clamp method, as voltage changes can be brought about easily. In contrast, in the FLIPR, it is not possible to induce voltage changes, and exactly because of the HCN activity, a hyperpolarization of the cells would only be transient. It has not been possible to achieve hyperpolarization of the transfected cells by adding an HCN2 inhibitor (zatebradine), since the resting membrane potential of the transfected cells is much too far removed from the potentials at which HCN2 is activated.

On the one hand, hyperpolarization is required for HCN activation. However, on the other hand, under physiological conditions, an activated HCN leads immediately to depolarization. Accordingly, in the present invention, conditions are provided under which the HCN channels can be activated by hyperpolarization, but where depolarization by the activated HCN channel is initially impossible. To this end, the cells, for example cells seeded in microtiter plates, are washed in an isoosmolar buffer in which NaCl has been replaced by another chloride salt, such as choline chloride. In embodiments, the wash buffer also contains at least some KCl, since extracellular K.sup.+ can improve HCN activation (Biel et al. 1999). In embodiments, the wash buffer contains at least 1 mM KCl. In embodiments, the wash buffer contains about 5 mM KCl. The wash buffer, which serves to effect hyperpolarization of the cation channels and thus the HCN cells, can also contain 5 .mu.M DiBac.sub.4 (3) for measuring changes in the membrane potential in the FLIPR. By removing the extracellular Na.sup.+, the cells are hyperpolarized, i.e. the cation channel is activated. However, the HCN is not capable of causing depolarization of the cells, since the required concentration gradient of the ions Na.sup.+ or K.sup.+ transported by HCN is missing. Here, an activated HCN could only result in a more pronounced hyperpolarization. This is reflected in the fact that the initial fluorescence measured for HCN cells in the FLIPR at 10 .mu.M forskolin is lower than that without forskolin, whereas there is no difference in nontransfected cells.

In the FLIPR, Na.sup.+ is added to the cells, so that the activated HCN (after a few seconds, in which there are mixing effects) causes, from about 15 seconds after the addition of Na.sup.+, depolarization of the cells, which becomes visible by an increase in fluorescence. The detection of HCN modulators can rely on a difference between cells having an activated HCN channel (e.g., only Na.sup.+ addition) and cells having a blocked HCN channel (e.g., Na.sup.+ +8 mM CsCl). It has been determined that a greater difference provides a greater reliability in the system. For example, activation of the HCN channel by pre-incubation with 10 .mu.M forskolin increases the difference between the uninhibited 100% value from the inhibited 0% value considerably.

One embodiment of the present invention relates to the comparative determination of the change in the membrane potential of at least two cell populations incubated with different concentrations of one of the substances to be examined. In this way, the optimal concentration of the substance(s) can be determined.

Substances that are to be examined for their activity are referred to as substances to be examined or substances to be tested. Substances that are active, i.e. that modulate the activity of the hyperpolarization-activated cation channel, can either be inhibitors (they inhibit the channel and reduce depolarization or prevent depolarization altogether) or be activators (they activate the channel and cause a more pronounced or more rapid depolarization) of the hyperpolarization-activated cation channel.

In embodiments, the invention provides a high-throughput screening (HTS) process. In HTS, the process can be used for identifying inhibitors and/or activators of a hyperpolarization-activated cation channel. Substances identified in this manner can be used, for example, as pharmaceutically active compounds. Thus, they can be used as medicaments (medicinal compositions) or as active ingredients of medicaments.

Accordingly, the invention also provides a process that comprises the formulation of an identified substance in a pharmaceutically acceptable form. In this aspect of the invention, the methods described above can be linked to formulation of an identified substance in a pharmaceutically acceptable form. Such forms, and processes for preparing such forms, are well known to, and widely practiced by, those of skill in the art. Therefore, they need not be detailed here. Examples include, but are not limited to, forms that comprise excipients or biologically tolerable carriers.

The invention also provides a process for preparing a medicament. The process comprises the identification of a substance that inhibits or activates the activity of a hyperpolarization-activated cation channel, and mixing the identified substance with a pharmaceutically acceptable excipient. In embodiments, the process for preparing a medicament comprises a) the identification of a substance which modulates the activity of hyperpolarization-activated cation channels; b) the preparation of the substance; c) the purification of the substance; and d) the mixing of the substance with a pharmaceutically acceptable excipient.

The invention also provides a kit. In embodiments, the kit is a test kit for determining whether a substance modulates the activity of a hyperpolarization-activated cation channel. In embodiments, the test kit comprises a) cells that overexpress a hyperpolarization-activated cation channel; b) an isoosmolar sodium-ion-free buffer for hyperpolarizing the cell; and c) at least one reagent for detection of hyperpolarization activated cation channels.

The components/reagents can be those described in detail herein with respect to the assays of the invention. The components can be supplied in separate containers within the kit or in combinations within containers within the kit. Where applicable, components and/or reagents can be supplied in stabilized form. The stabilized form can permit the components and/or reagents to be maintained for extended periods of time without significant degradation or loss in activity. For example, the cells can be supplied in a cryogenic state. In addition, the salts (ions) or reagents that will comprise the assay composition can be provided in solid (dry) form, to be reconstituted with water or another appropriate solvent prior to use. Accordingly, the kit can comprise water.

As a measure for the activity of a substance, the change in the membrane potential of the cell is measured, for example, with the aid of a potential-sensitive fluorescent dye. As mentioned above, the dye can be an oxanol derivative, such as 3-bis-barbituric acid oxanol.

EXAMPLES

The invention will now be illustrated in more detail by various examples of embodiments of the invention. The following examples are exemplary only. Thus, the scope of the invention is not limited to the embodiments disclosed in the examples. Abbreviations used in the Examples are listed in Table 7 below.

Example 1

Preparation of Transfected Cells

The plasmid pcDNA3-muHCN2 contains the murine HCN2 (muHCN2) cDNA (Genbank Accession No. AJ225122) of Pos. 22-2812 (coding sequence: Pos. 36-2627), cloned into the EcoRI and NotI cleavage sites of pcDNA3, and was obtained from M. Biel, TU Munich (Ludwig et al., 1998). In each case 6 .mu.g of this plasmid DNA were used for transfecting CHO or HEK293 cells. For transfecting CHO cells or HEK cells, the LipofectAmine.TM. Reagent from Life Technologies (Gaithersburg, Md., USA) was used, in accordance with the instructions of the manufacturer. 24 hours after the transfection, the cells were transferred from culture dishes into 75 cm.sup.2 cell culture bottles. 72 hours after the transfection, the cells were subjected to a selection with 400 .mu.g/ml of the antibiotic G418 (Calbiochem, Bad Soden, Germany). Following a two-week selection, the surviving cells were detached from the bottles using trypsin-EDTA, counted in the cell counter Coulter Counter Z1 and sown into 96-well microtiter plates such that statistically, 1 cell was present per well. The microtiter plates were checked regularly under the microscope, and only cells from wells in which only one colony was growing were cultured further.

From these cells, total RNA was isolated with the aid of the QlAshredder and RNeasy kits from Qiagen (Hilden, Germany). This total RNA was examined by RT-PCR for expression of muHCN2 (Primer 1): 5'-GCCAATACCAGGAGAAG-3' [SEQ ID NO. 7], corresponds to Pos. 1354-1370 and AJ225122, and primer 2:5'-TGAGTAGAGGCGACAGTAG-3' [SEQ ID NO. 8], corresponds to pos. 1829-1811 in AJ225122; expected RT-PCR band: 476 bp.

Example 2

Patch-Clamp Examination of the Cells

Using the patch-clamp method, the cells with detectable mRNA expression were examined electrophysiologically, in the whole-cell configuration, for functional expression of muHCN2. This method is described in detail in Hamill et al (1981), which is incorporated herein by reference. The cells were clamped to a holding potential of -40 mV. Starting with this holding potential, the ion channels were activated by a voltage change to B140 mV for a period of one second. The current amplitude was determined the end of this pulse. Among the transfected HEK cells, some were found having currents of about 1 nA; however, owing to interfering endogenous currents, it was not possible to construct an assay for these cells in the FLIPR.

However, in the HEK cells, it was found clearly that a functionally active HCN2 channel was only detectable in cells having strong mRNA expression. In the CHO cells, the correlation between mRNA expression and function was confirmed. In general, the mRNA expression in the HEK cells was about three times better than that in the CHO cells. In the patch-clamp studies, it was possible to demonstrate a weak current in some cells of one of the most strongly expressing CHO cell lines.

Example 3

Preparation of Doubly-Transfected Cells

Since the functional expression appeared to correlate strongly with the mRNA expression, we carried out a second transfection with the muHCN2 cDNA that had earlier been cloned into the EcoRI and NotI site of the vector pcDNA3.1(+)zeo. After a two-week selection with G418 and Zeocin (Invitrogen, Groningen, NL), individual cell clones were isolated as described in Example 1. Following isolation of the total RNA from these cells, an RT-PCR with the primers mentioned in Example 1 was carried out. Then an RT-PCR was carried out with the following primers, comprising a region which contains the 3'-end of the coding sequence of muHAC1 (primer 3: 5'-AGTGGCCTCGACCCACTGGACTCT-3' [SEQ ID NO. 9], corresponds to pos. 2553-2576 in AJ225122, and primer 4: 5'-CCGCCTCCTAAGCTACCTACGTCCC-3' [SEQ ID NO. 10], corresponds to pos. 2725-2701 in AJ225122).

Some of the doubly-transfected cells showed a considerably more pronounced expression both in RT-PCR and in the patch-clamp analysis than the cells which had been transfected only once. Electrophysiologically, currents of up to 11 nA were measured. These cells were used for constructing an FLIPR assay for HCN2.

Example 4

Construction of an FLIPR Assay for HCN Channels

The cells seeded on the microtiter plates are washed in an isoosmolar buffer in which NaCl has been replaced by choline chloride. However, this wash buffer also contains 5 mM KCl, since extracellular K.sup.+ is important for HCN activation (Biel et al. 1999). This wash buffer, which serves to effect hyperpolarization of the HCN cells, also contains 5 .mu.M DiBac.sub.4 (3) for measuring changes in the membrane potential in the FLIPR. By removing the extracellular Na.sup.+, the cells are hyperpolarized, i.e. the HCN is activated. However, the HCN is not capable of causing depolarization of the cells, since the required concentration gradient of the ions Na.sup.+ or K.sup.+ transported by HCN is missing. Here, an activated HCN could only result in a more pronounced hyperpolarization. This is reflected in the fact that the initial fluorescence measured for HCN cells in the FLIPR at 10 .mu.M forskolin is lower than that without forskolin, whereas there is no difference in nontransfected cells.

Since DiBac.sub.4 (3) fluorescence may be sensitive to temperature variations, the measurement is, after an incubation at 37.degree. C. for 30 minutes, carried out as quickly as possible--cooling of the dye solution may affect the measured results. Preferably, the sample is thermostated for five minutes in the FLIPR prior to the start of the measurement.

The substances to be tested are preferably added in 10-fold concentrated form to the cells which had been dyed with DiBac.sub.4 (3).

In the FLLPR, Na.sup.+ is added to the cells so that the activated HCN (after a few seconds, in which there are mixing effects) causes, from about 15 seconds after the addition of Na.sup.+, depolarization of the cells, which becomes visible by an increase in fluorescence. An activation of the HCN channel by preincubation with 10 .mu.M forskolin increases the difference between the uninhibited 100% value from the inhibited 0% value considerably. By comparison with the control values, it can be detected whether a substance to be tested is an activator (more rapid or more pronounced depolarization) or an inhibitor (slower or inhibited depolarization).

Example 5

Determination of the IC50 of an HCN2 Blocker

Using the transfected HCN cells, the effect of various concentrations of the substance zatebradine, which is known as an I.sub.f blocker, were examined. The inhibition by zatebradine was calculated from the relative change in fluorescence from the time 60 seconds. For each concentration of the inhibitor, the mean of in each case 6 wells of the microtiter plate was determined. From these values, the IC50 of zatebradine was calculated as 26 .mu.M, a value which corresponds well with the value of 31 .mu.M determined electrophysiologically in the same cells.

Example 6

Use of the FLIPR Membrane Assay Kit (Molecular Devices, Sunnyvale, USA):

Cells that were seeded a day earlier are, as before, washed three times with in each case 400 .mu.l of wash buffer per well. However, this time, the volume that remains above the cells after the last washing step is chosen depending on the desired Na.sup.+ and Cs.sup.+ concentrations. The dye, in wash buffer, is added, and the cells are incubated with dye for 30 minutes. The temperature is typically room temperature (about 21-25.degree. C.), but can be about 37.degree. C.

In the FLIPR, depolarization is then induced by addition of Na.sup.+ and in some control wells inhibited again by simultaneous addition of Cs.sup.+. Since, in the dye from Molecular Devices, an increase in the ionic strength might lead to changes in fluorescence, it has to be ensured that the ionic strength changes to the same degree in all wells of a microtiter plate. The desired final concentrations of sodium or cesium ions permitting, the osmolarity is not changed. To adjust the desired concentrations of Na.sup.+ and Cs.sup.+, two further buffers which, instead of 140 mM of choline chloride, contain 140 mM NaCl (sodium buffer) and 140 mM CsCl (cesium buffer), respectively, are used in addition to the wash buffer.

For measurements with the FLIPR Membrane Potential Assay Kit Molecular Devices gives the following standard protocol for 96-well microtiter plates (384 wells in brackets): On the day before the measurement, the cells are seeded in 100 ml (25 ml) of medium. Following addition of 100 .mu.l (25 .mu.l) of dye and 30 minutes of incubation at room temperature or at 37.degree. C., 50 .mu.l (25 .mu.l) of the substance to be tested, in a suitable buffer, are added in the FLIPR.

Using the volumes stated by Molecular Devices, it is possible, without changing the ionic strength, to achieve a maximum concentration of 28 mM for Na.sup.+ +Cs.sup.+ in 96-well plates and a maximum concentration of 46.7 mM in 384-well plates. Since this concentration, in particular in the 96-well plates, is too low for optimum activity of the hyperpolarization-activated cation channels, different volumes are tested for the individual steps.

It has been found that the dye concentrations can be reduced to half of those in the protocol given by Molecular Devices.

In 96-well plates, good results are obtained even with the following volumes: 45 .mu.l of wash buffer supernatant above the cells, 60 .mu.l of dye in the wash buffer, 195 .mu.l addition volume in the FLIPR. Such a high additional volume allows a maximum concentration of Na.sup.+ +Cs.sup.+ of 91 mM, i.e. at 8-10 mM CsCl, the final NaCl concentration can be 81-83 mM. For 80 mM Na.sup.+ and 8 mM Cs.sup.+, 6.43 .mu.l of wash buffer, 171.43 .mu.l of sodium buffer and 17.14 .mu.l of cesium buffer are required, based on an added volume of 195 .mu.l.

Materials and Methods

The following materials and methods were, and can be, used to practice the invention as described in the Examples above. Other materials and methods can be used to practice other embodiments of the invention. Thus, the invention is not limited to the materials and methods disclosed below.

1. Solutions and buffers for the measurement with DiBac.sub.4 (3) A: DiBac.sub.4 (3) bis-(1,3-dibutylbarbituric acid)trimethine oxonol From Molecular Probes, Cat. No. B-438, MW: 516.64 g/mol

A 10 mM stock solution of DiBac.sub.4 (3) is made up in DMSO (25 mg of DiBac.sub.4 (3)/4.838 ml of DMSO). Aliquots of this stock solution are stored at -20.degree. C. Final concentration during dyeing and addition: 5 .mu.M. B: Forskolin MW: 410.5 g/mol Final concentration during dyeing: 10 .mu.M Aliquots of a 10 mM stock solution in DMSO are stored at -20.degree. C. C: Wash buffer: (140 mM choline chloride, 5 mM KCl, 1 mM CaCl.sub.2, 1 mM MgCl.sub.2, 10 mM HEPES, 5 mM glucose, adjusted to pH 7.4 with 1 M KOH) D: Presoak solution for saturating the tips of the pipettes: as wash buffer+10 .mu.M DiBac.sub.4 (3) This solution is only used for the presoak plate. E: Dye solution: double concentrated, i.e. wash buffer+10 .mu.M DiBac.sub.4 (3)+20 .mu.M forskolin F: 10-fold concentrated solution for the addition plate: 500 mM NaCl in H.sub.2 O+5 .mu.M DiBac.sub.4 (3) All substances are made up in this solution in 10-fold concentrated form. Positive control (final concentration): 50 mM NaCl Negative control (final concentration): 50 mM NaCl+8 mM CsCl

2. Solutions and buffers for the measurements with the FLIPR Membrane Potential Assay Kit from Molecular Devices A: FLIPR Membrane Potential Assay Kit, from Molecular Probes, Cat. No. R8034 B: Wash buffer: (140 mM choline chloride, 5 mM KCl, 1 mM CaCl.sub.2, 1 mM MgCl.sub.2, 10 mM HEPES, 5 mM glucose, adjusted to pH 7.4 with 1M KOH). C: Dye buffer: (content of one of the "reagent vials" of the FLIPR Membrane Potential Assay Kit in 10 ml of wash buffer) D: Sodium buffer: (140 mM NaCl, 5 mM KCl, 1 mM CaCl.sub.2, 1 mM MgCl.sub.2, 10 mM HEPES, 5 mM glucose, adjusted to pH 7.4 with 1M KOH). E: Cesium buffer: (140 mM CsCl, 5 mM KCl, 1 mM CaCl.sub.2, 1 mM MgCl.sub.2, 10 mM HEPES, 5 mM glucose, adjusted to pH 7.4 with 1M KOH).

3. Cell culture operations:

The day before the measurement, the muHCN2-transfected CHO cells are seeded at a density of 35 000 cells/well, in each case in 200 .mu.l of complete medium, into black 96-well microtiter plates. The cells are incubated at 37.degree. C. and 5% CO.sub.2 overnight.

4. Dyeing with DiBac.sub.4 (3) and measurement in FLIPR:

Before dyeing, the cells are washed three times with 400 .mu.l of wash buffer in a cell washer. After the last washing step, a residual volume of 90 .mu.l of wash buffer/well remains above the cells.

The washed cells (with 90 .mu.l of wash buffer/well) are in each case incubated with 90 .mu.l of dye solution/well at 37.degree. C. in the CO.sub.2 incubator for 30 minutes. After this incubation time, the cell plate is measured in the FLIPR at about 37.degree. C. (preset temperature setting of the FLIPR manufacturer for measurement of membrane potentials with DiBac.sub.4 (3)), either immediately or after five minutes of thermostating.

The snapshot (initial fluorescence before the start of the measurement) should on average be about 35 000 units. In the maximum, the FLIPR can resolve up to about 65 000 units.

When the program is started, the tips of the pipettes are initially saturated by immersion into presoak solution with DiBac.sub.4 (3). Following this step, the actual measurement is initiated with the first measurement (t=0 seconds). Since DiBac.sub.4 (3) is a slow-response dye, it is sufficient to determine the fluorescence in the wells of the microtiter plate every 5 seconds. After about 20 seconds, the substances, which are present in the addition plate in 10-fold concentrated form, are added simultaneously to the microtiter plate using the pipettor. Since the volume after dyeing is 180 .mu.l, 20 .mu.l are added to each well. The measurement of the fluorescence can be terminated after about 5 minutes. For evaluation, the change in fluorescence in the interval where it is linear and in which uninhibited HCN2-transfected cells differ significantly from inhibited cells is examined.

5. Dyeing with the FLIPR Membrane Potential Assay Kit and measurement in the FLIPR.

Before dyeing, the cells are washed three times with 400 .mu.l of wash buffer in a cell washer. After the last washing step, a residual volume of 45-90 .mu.l of wash buffer/well remains above the cells.

Following addition of the dye solution (the volume depends on the desired final concentrations), the samples are incubated at room temperature (preferred) or at 37.degree. C. in a CO.sub.2 incubator for 30 minutes. Following this incubation time, the cell plate is measured at room temperature in the FLIPR.

In the FLIPR Membrane Potential Assay Kit the snapshot (initial fluorescence before the start of the measurement) may be lower than that during the measurement with DiBac.sub.4 (3), since the assay kit is more sensitive to changes in the membrane potential than DiBac.sub.4 (3).

Owing to the higher achievable sensitivity, the measurement should, wherever possible (FLIPRII), be carried out using an emission filter which is transparent to light above 550 nm. However, it is also possible to carry out the measurements using the standard filter, which is transparent between 510 and 580 nm.

When the program is started (t=0), the FLIPR initially determines the fluorescence of all wells of the plate a number of times, before the depolarization is started after about 20 seconds by addition of sodium ions. In each case, the addition solution is mixed from the three buffers (wash buffer, sodium buffer and cesium buffer) such that the addition results in no change of the osmolarity, or in a change which is identical in all wells. The measurement of the fluorescence can be terminated after about 5 minutes. The wells to which, in addition to Na.sup.+, 8 mM Cs.sup.+ were added to block the HCN channel completely serve as negative control. By deducting these values from the others, a good measure for the activity of the HCN channel under the influence of the substance to be examined is obtained. For evaluation, the change in fluorescence in the interval where it is linear and in which uninhibited HCN2-transfected cells differ significantly from inhibited cells is examined.

REFERENCES All references disclosed herein, including the following references, are hereby incorporated herein by reference. Biel M., Ludwig A., Zong X., Hofmann R. (1999) Hyperpolarization-activated cation channels: A multigene family. Rev. Physiol. Biochem. Pharmacol. 136: 165-181. Hamill O. P., Marty A., Neher E., Sakmann B., Sigworth F. J. (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 391: 85-100. Ludwig A., Zong X., Jeglitsch M., Hofmann F., Biel M. (1998) A family of hyperpolarization-activated mammalian cation channels. Nature 393: 587-591. Ludwig A., Zong X., Stieber J., Hullin R., Hofmann R., Biel M. (1999) Two pacemaker channels from heart with profoundly different activation kinetics. EMBO J. 18: 2323-2329. Reiffen A., Eberlein W., Muller P., Psiorz M., Noll K., Heider J., Lillie C., Kobinger W., Luger P. (1990) Specific bradycardiac agents. 1. Chemistry, pharmacology, and structure-activity relationships of substituted benzazepinones, a new class of compounds exerting antiischemic properties. J. Med. Chem. 33: 1496-1504.

TABLE 1 SEQ ID NO.1 Protein sequence of huHCN2 Accession number: AAC28444 1 MDARGGGGRP GESPGASPTT GPPPPPPPRP PKQQPPPPPP PAPPPGPGPA PPQHPPRAEA 61 LPPEAADEGG PRGRLRSRDS SCGRPGTPGA ASTAKGSPNG ECGRGEPQCS PAGFEGPARG 121 PKVSFSCRGA ASGPAPGPGP AEEAGSEEAG PAGEPRGSQA SFMQRQFGAL LQPGVNKFSL 181 RMFGSQKAVE REQERVKSAG AWIIHPYSDF RFYWDFTMLL FMVGNLIIIP VGITFFKDET 241 TAPWIVFNVV SDTFFLMDLV LNFRTGIVIE DNTEIILDPE KIKKKYLRTW FVVDFVSSIP 301 VDYIFLIVEK GIDSEVYKTA RALRIVRFTK ILSLLRLLRL SRLIRYIHQW EEIFRMTYDL 361 ASAVMRICNL ISMMLLLCHW DGCLQFLVPM LQDFPRNCWV SINGMVNRSW SELYSFALFK 421 AMSHMLCIGY GRQAPESMTD IWLTMLSMIV GATCYAMFIG HATALIQSLD SSRRQYQEKY 481 KQVEQYMSFH KLPADFRQKI HDYYEHRYQG KMFDEDSILG ELNGPLREEI VNFNCRKLVA 541 SMPLFANADP NFVTAMLTKL KFEVFQPGDY IIREGTIGKK MYFIQHGVVS VLTKGNKEMK 601 LSDGSYFGEI CLLTRGRRTA SVRADTYCRL YSLSVDNFNE VLEEYPMMRR AFETVAIDRL 661 DRIGKKNSIL LHKVQHDLNS GVFNNQENAI IQEIVKYDRE MVQQAELGQR VGLFPPPPPP 721 PQVTSAIATL QQAAAMSFCP QVARPLVGPL ALGSPRLVRR PPPGPAPAAA SPGPPPPASP 781 PGAPASPRAP RTSPYGGLPA APLAGPALPA RRLSRASRPL SASQPSLPHG APGPAASTRP 841 ASSSTPRLGP TPAARAAAPS PDRRDSASPG AAGGLDPQDS ARSRLSSNL

TABLE 2 SEQ ID NO.2 Nucleotide sequence of huHCN2 Accession number: AF065164 1 CGGCTCCGCT CCGCACTGCC CGGCGCCGCC TCGCCATGGA CGCGCGCGGG GGCGGCGGGC 61 GGCCCGGGGA GAGCCCGGGC GCGAGCCCCA CGACCGGGCC GCCGCCGCCG CCGCCCCCGC 121 GCCCCCCCAA ACAGCAGCCG CCGCCGCCGC CGCCGCCCGC GCCCCCCCCG GGCCCCGGGC 181 CCGCGCCCCC CCAGCACCCG CCCCGGGCCG AGGCGTTGCC CCCGGAGGCG GCGGATGAGG 241 GCGGCCCGCG GGGCCGGCTC CGCAGCCGCG ACAGCTCGTG CGGCCGCCCC GGCACCCCGG 301 GCGCGGCGAG CACGGCCAAG GGCAGCCCGA ACGGCGAGTG CGGGCGCGGC GAGCCGCAGT 361 GCAGCCCCGC GGGGCCCGAG GGCCCGGCGC GGGGGCCCAA GGTGTCGTTC TCGTGCCGCG 421 GGGCGGCCTC GGGGCCCGCG CCGGGGCCGG GGCCGGCGGA GGAGGCGCGC AGCGAGGAGG 481 CGGGCCCGGC GGGGGAGCCG CGCGGCAGCC AGGCCAGCTT CATGCAGCGC CAGTTCGGCG 541 CGCTCCTGCA GCCGGGCGTC AACAAGTTCT CGCTGCGGAT GTTCGGCAGC CAGAAGGCCG 601 TGGAGCGCGA GCAGGAGCGC GTCAAGTCGG CGGGGGCCTG GATCATCCAC CCGTACAGCG 661 ACTTCAGGTT CTACTGGGAC TTCACCATGC TGCTGTTCAT GGTGGGAAAC CTCATCATCA 721 TCCCAGTGGG CATCACCTTC TTCAAGGATG AGACCACTGC CCCGTGGATC GTGTTCAACG 781 TGGTCTCGGA CACCTTCTTC CTCATGGACC TGGTGTTGAA CTTCCGCACC GGCATTGTGA 841 TCGAGGACAA CACGGAGATC ATCCTGGACC CCGAGAAGAT CAAGAAGAAG TATCTGCGCA 901 CGTGGTTCGT GGTGGACTTC GTGTCCTCCA TCCCCGTGGA CTACATCTTC CTTATCGTGG 961 AGAAGGGCAT TGACTCCGAG GTCTACAAGA CGGCACGCGC CCTGCGCATC GTGCGCTTCA 1021 CCAAGATCCT CAGCCTCCTG CGGCTGCTGC GCCTCTCACG CCTGATCCGC TACATCCATC 1081 AGTGGGAGGA GATCTTCCAC ATGACCTATG ACCTGGCCAG CGCGGTGATG AGGATCTGCA 1141 ATCTCATCAG CATGATGCTG CTGCTCTGCC ACTGGGACGG CTGCCTGCAG TTCCTGGTGC 1201 CTATGCTGCA GGACTTCCCG CGCAACTGCT GGGTGTCCAT CAATGGCATG GTGAACCACT 1261 CGTGGAGTGA ACTGTACTCC TTCGCACTCT TCAAGGCCAT GAGCCACATG CTGTGCATCG 1321 GGTACGGCCG GCAGGCGCCC GAGAGCATGA CGGACATCTG GCTGACCATG CTCAGCATGA 1381 TTGTGGGTGC CACCTGCTAC GCCATGTTCA TCGGCCACGC CACTGCCCTC ATCCAGTCGC 1441 TGGACTCCTC GCGGCGCCAG TACCAGGAGA AGTACAAGCA GGTGGAGCAG TACATGTCCT 1501 TCCACAAGCT GCCAGCTGAC TTCCGCCAGA AGATCCACGA CTACTATGAG CACCGTTACC 1561 AGGGCAAGAT GTTTGACGAG GACAGCATCC TGGGCGAGCT CAACGGGCCC CTGCGGGAGG 1621 AGATCGTCAA CTTCAACTGC CGGAAGCTGG TGGCCTCCAT GCCGCTGTTC GCCAACGCCG 1681 ACCCCAACTT CGTCACGGCC ATGCTGACCA AGCTCAAGTT CGAGGTCTTC CAGCCGGGTG 1741 ACTACATCAT CCGCGAAGGC ACCATCGGGA AGAAGATGTA CTTCATCCAG CACGGCGTGG 1801 TCAGCGTGCT CACTAAGGGC AACAAGGAGA TGAAGCTGTC CGATGGCTCC TACTTCGGGG 1861 AGATCTGCCT GCTCACCCGG GGCCGCCGCA CGGCGAGCGT GCGGGCTGAC ACCTACTGCC 1921 GCCTCTATTC GCTGAGCGTG GACAACTTCA ACGAGGTGCT GGAGGAGTAC CCCATGATGC 1981 GGCGCGCCTT CGAGACGGTG GCCATCGACC GCCTGGACCG CATCGGCAAG AAGAATTCCA 2041 TCCTCCTGCA CAAGGTGCAG CATGACCTCA ACTCGGGCGT ATTCAACAAC CAGGAGAACG 2101 CCATCATCCA GGAGATCGTC AAGTACGACC GCGAGATGGT GCAGCAGGCC GAGCTGGGTC 2161 AGCGCGTGGG CCTCTTCCCG CCGCCGCCGC CGCCGCCGCA GGTCACCTCG GCCATCGCCA 2221 CGCTGCAGCA GGCGGCGGCC ATGAGCTTCT GCCCGCAGGT GGCGCGGCCG CTCGTGGGGC 2281 CGCTGGCGCT CGGCTCGCCG CGCCTCGTGC GCCGCCCGCC CCCGGGGCCC GCACCTGCCG 2341 CCGCCTCACC CGGGCCCCCG CCCCCCGCCA GCCCCCCGGG CGCGCCCGCC AGCCCCCGGG 2401 CACCGCGGAC CTCGCCCTAC GGCGGCCTGC CCGCCGCCCC CCTTGCTGGG CCCGCCCTGC 2461 CCGCGCGCCG CCTGAGCCGC GCGTCGCGCC CACTGTCCGC CTCGCAGCCC TCGCTGCCTC 2521 ACGGCGCCCC CGGCCCCGCG GCCTCCACAC GCCCGGCCAG CAGCTCCACA CCGCGCTTGG 2581 GGCCCACGCC CGCTGCCCGG GCCGCCGCGC CCAGCCCGGA CCGCAGGGAC TCGGCCTCAC 2641 CCGGCGCCGC CGGCGGCCTG GACCCCCAGG ACTCCGCGCG CTCGCGCCTC TCGTCCAACT 2701 TGTGACCCTC GCCGACCGCC CCGCGGGCCC AGGCGGGCCG GGGGCGGGGC CGTCATCCAG 2761 ACCAAAGCCA TGCCATTGCG CTGCCCCGGC CGCCAGTCCG CCCAGAAGCC ATAGACGAGA 2821 CGTAGGTAGC CGTAGTTGGA CGGACGGGCA GGGCCGGCGG GGCAGCCCCC TCCGCGCCCC 2881 CGGCCGTCCC CCCTCATCGC CCCGCGCCCA CCCCCATCGC CCCTGCCCCC GGCGGCGGCC 2941 TCGCGTGCGA GGGGGCTCCC TTCACCTCGG TGCCTCAGTT CCCCCAGCTC TAAGACAGGG 3001 ACGGGGCGGC CCAGTGGCTG AGAGGAGCCG GCTGTGGAGC CCCGCCCGCC CCCCACCCTC 3061 TAGGTGGCCC CCGTCCGAGG AGGATCGTTT TCTAAGTGCA ATACTTGGCC CGCCGGCTTC 3121 CCGCTGCCCC CATCGCGCTC ACGCAATAAC CGGCCCGGCC CCCGTCCGCG CGCGTCCCCC 3181 GGTGACCTCG GGGAGCAGCA CCCCGCCTCC CTCCAGCACT GGCACCGAGA GGCAGGCCTG 3241 GCTGCGCAGG GCGCGGGGGG GAGGCTGGGG TCCCGCCGCC GTGATGAATG TACTGACGAG 3301 CCGAGGCAGC AGTGCCCCCA CCGTGGCCCC CCACGCCCCA TTAACCCCCA CACCCCCATT 3361 CCGCGCAATA AA

TABLE 3 SEQ ID NO.3 Protein sequence of huHCN4 Accession number: HSA132429 1 MDKLPPSMRK RLYSLPQQVG AKAWIMDEEE DAEEEGAGGR QDPSRRSIRL 51 RPLPSPSPSA AAGGTESRSS ALGAADSEGP ARGAGKSSTN GDCRRFRGSL 101 ASLGSRGGGS GGTGSGSSHG HLHDSAEERR LIAEGDASPG EDRTPPGLAA 151 EPERPGASAQ PAASPPPPQQ PPQPASASCE QPSVDTAIKV EGGAAAGDQI 201 LPEAEVRLGQ AGFMQRQFGA MLQPGVNKFS LRMFGSQKAV EREQERVKSA 251 GFWIIHPYSD FRFYWDLTML LLMVGNLIII PVGITFFKDE NTTPWIVFNV 301 VSDTFFLIDL VLNFRTGIVV EDNTEIILDP QRIKMKYLKS WFMVDFISSI 351 PVDYIFLIVE TRIDSEVYKT ARALRIVRFT KILSLLRLLR LSRLIRYIHQ 401 WEEIFHMTYD LASAVVRIVN LIGMMLLLCH WDGCLQFLVP MLQDFPDDCW 451 VSINNMVNNS WGKQYSYALF KAMSHMLCIG YGRQAPVGMS DVWLTMLSMI 501 VGATCYAMFI GHATALIQSL DSSRRQYQEK YKQVEQYMSF HKLPPDTRQR 551 IHDYYEHRYQ GKMFDEESIL GELSEPLREE IINFNCRKLV ASMPLFANAD 601 PNFVTSMLTK LRFEVFQPGD YIIREGTIGK KMYFIQHGVV SVLTKGNKET 651 KLADGSYFGE ICLLTRGRRT ASVRADTYCR LYSLSVDNFN EVLEEYPMMR 701 RAFETVALDR LDRIGKKNSI LLHKVQHDLN SGVFNYQENE IIQQIVQHDR 751 EMAHCAHRVQ AAASATPTPT PVIWTPLIQA PLQAAAATTS VAIALTHHPR 801 LPAAIFRPPP GSGLGNLGAG QTPRHLKRLQ SLIPSALGSA SPASSPSQVD 851 TPSSSSFHIQ QLAGFSAPAG LSPLLPSSSS SPPPGACGSP SAPTPSAGVA 901 ATTIAGFGHF HKALGGSLSS SDSPLLTPLQ PGARSPQAAQ PSPAPPGARG 951 GLGLPEHFLP PPPSSRSPSS SPGQLGQPPG ELSLGLATGP LSTPETPPRQ 1001 PEPPSLVAGA SGGASPVGFT PRGGLSPPGH SPGPPRTFPS APPRASGSHG 1051 SLLLPPASSP PPPQVPQRRG TPPLTPGRLT QDLKLISASQ PALPQDGAQT 1101 LRRASPHSSG ESMAAFPLFP RAGGGSGGSG SSGGLGPPGR PYGAIPGQHV 1151 TLPRKTSSGS LPPPLSLFGA RATSSGGPPL TAGPQREPGA RPEPVRSKLP 1201 SNL*

TABLE 4 SEQ ID NO.4 Nucleotide sequence of huHCN4 Accession number: HSA132429 1 GGTCGCTGGG CTCCGCTCGG TTGCGGCGGG AGCCCCGGGA CGGGCCGGAC GGGCCGGGGC 61 AGAGGAGGCG AGGCGAGCTC GCGGGTGGCC AGCCACAAAG CCCGGGCGGC GAGACAGACG 121 GACAGCCAGC CCTCCCGCGG GACGCACGCC CGGGACCCGC GCGGGCCGTG CGCTCTGCAC 181 TCCGGAGCGG TTCCCTGAGC GCCGCGGCCG CAGAGCCTCT CCGGCCGGCG CCCATTGTTC 241 CCCGCGGGGG CGGGGCGCCT GGAGCCGGGC GGCGCGCCGC GCCCCTGAAC GCCAGAGGGA 301 GGGAGGGAGG CAAGAAGGGA GCGCGGGGTC CCCGCGCCCA GCCGGGCCCG GGAGGAGGTG 361 TAGCGCGGCG AGCCCGGGGA CTCGGAGCGG GACTAGGATC CTCCCCGCGG CGCGCAGCCT 421 GCCCAAGCAT GGGCGCCTGA GGCTGCCCCC ACGCCGGCGG CAAAGGACGC GTCCCCACGG 481 GCGGACTGAC CGGCGGGCGG ACCTGGAGCC CGTCCGCGGC GCCGCGCTCC TGCCCCCGGC 541 CCGGTCCGAC CCCGGCCCCT GGCGCCATGG ACAAGCTGCC GCCGTCCATG CGCAAGCGGC 601 TCTACAGCCT CCCGCAGCAG GTGGGGGCCA AGGCGTGGAT CATGGACGAG GAAGAGGACG 661 CCGAGGAGGA GGGGGCCGGG GGCCGCCAAG ACCCCAGCCG CAGGAGCATC CGGCTGCGGC 721 CACTGCCCTC GCCCTCCCCC TCGGCGGCCG CGGGTGGCAC GGAGTCCCGG AGCTCGGCCC 781 TCGGGGCAGC GGACAGCGAA GGGCCGGCCC GCGGCGCGGG CAAGTCCAGC ACGAACGGCG 841 ACTGCAGGCG CTTCCGCGGG AGCCTGGCCT CGCTGGGCAG CCGGGGCGGC GGCAGCGGCG 901 GCACGGGGAG CGGCAGCAGT CACGGACACC TGCATGACTC CGCGGAGGAG CGGCGGCTCA 961 TCGCCGAGGG CGACGCGTCC CCCGGCGAGG ACAGGACGCC CCCAGGCCTG GCGGCCGAGC 1021 CCGAGCGCCC CGGCGCCTCG GCGCAGCCCG CAGCCTCGCC GCCGCCGCCC CAGCAGCCAC 1081 CGCAGCCGGC CTCCGCCTCC TGCGAGCAGC CCTCGGTGGA CACCGCTATC AAAGTGGAGG 1141 GAGGCGCGGC TGCCGGCGAC CAGATCCTCC CGGAGGCCGA GGTGCGCCTG GGCCAGGCCG 1201 GCTTCATGCA GCGCCAGTTC GGGGCCATGC TCCAACCCGG GGTCAACAAA TTCTCCCTAA 1261 GGATGTTCGG CAGCCAGAAA GCCGTGGAGC GCGAACAGGA GAGGGTCAAG TCGGCCGGAT 1321 TTTGGATTAT CCACCCCTAC AGTGACTTCA GATTTTACTG GGACCTGACC ATGCTGCTGC 1381 TGATGGTGGG AAACCTGATT ATCATTCCTG TGGGCATCAC CTTCTTCAAG GATGAGAACA 1441 CCACACCCTG GATTGTCTTC AATGTGGTGT CAGACACATT CTTCCTCATC GACTTGGTCC 1501 TCAACTTCCG CACAGGGATC GTGGTGGAGG ACAACACAGA GATCATCCTG GACCCGCAGC 1561 GGATTAAAAT GAAGTACCTG AAAAGCTGGT TCATGGTAGA TTTCATTTCC TCCATCCCCG 1621 TGGACTACAT CTTCCTCATT GTGGAGACAC GCATCGACTC GGAGGTCTAC AAGACTGCCC 1681 GGGCCCTGCG CATTGTCCGC TTCACGAAGA TCCTCAGCCT CTTACGCCTG TTACGCCTCT 1741 CCCGCCTCAT TCGATATATT CACCAGTGGG AAGAGATCTT CCACATGACC TACGACCTGG 1801 CCAGCGCCGT GGTGCGCATC GTGAACCTCA TCGGCATGAT GCTCCTGCTC TGCCACTGGG 1861 ACGGCTGCCT GCAGTTCCTG GTACCCATGC TACAGGACTT CCCTGACGAC TGCTGGGTGT 1921 CCATCAACAA CATGGTGAAC AACTCCTGGG GGAAGCAGTA CTCCTACGCG CTCTTCAAGG 1981 CCATGAGCCA CATGCTGTGC ATCGGCTACG GGCGGCAGGC GCCCGTGGGC ATGTCCGACG 2041 TCTGGCTCAC CATGCTCAGC ATGATCGTGG GTGCCACCTG CTACGCCATG TTCATTGGCC 2101 ACGCCACTGC CCTCATCCAG TCCCTGGACT CCTCCCGGCG CCAGTACCAG CAAAAGTACA 2161 AGCAGGTGGA GCAGTACATG TCCTTTCACA AGCTCCCGCC CGACACCCGG CAGCGCATCC 2221 ACGACTACTA CGAGCACCGC TACCAGGGCA AGATGTTCGA CGAGGAGAGC ATCCTGGGCG 2281 AGCTAAGCGA GCCCCTGCGG GAGGAGATCA TCAACTTTAA CTGTCGGAAG CTGGTGGCCT 2341 CCATGCCACT GTTTGCCAAT GCGGACCCCA ACTTCGTGAC GTCCATGCTG ACCAAGCTGC 2401 GTTTCGAGGT CTTCCAGCCT GGGGACTACA TCATCCGGGA AGGCACCATT GGCAAGAAGA 2461 TGTACTTCAT CCAGCATGGC GTGGTCAGCG TGCTCACCAA GGGCAACAAG GAGACCAAGC 2521 TGGCCGACGG CTCCTACTTT GGAGAGATCT GCCTGCTGAC CCGGGGCCGG CGCACAGCCA 2581 GCGTGAGGGC CGACACCTAC TGCCGCCTCT ACTCGCTGAG CGTGGACAAC TTCAATGAGG 2641 TGCTGGAGGA GTACCCCATG ATGCGAAGGG CCTTCGAGAC CGTGGCGCTG GACCGCCTGG 2701 ACCGCATTGG CAAGAAGAAC TCCATCCTCC TCCACAAAGT CCAGCACGAC CTCAACTCCG 2761 GCGTCTTCAA CTACCAGGAG AATGAGATCA TCCAGCAGAT TGTGCAGCAT GACCGGGAGA 2821 TGGCCCACTG CGCGCACCGC GTCCAGGCTG CTGCCTCTGC CACCCCAACC CCCACGCCCG 2881 TCATCTGGAC CCCGCTGATC CAGGCACCAC TGCAGGCTGC CGCTGCCACC ACTTCTGTGG 2941 CCATAGCCCT CACCCACCAC CCTCGCCTGC CTGCTGCCAT CTTCCGCCCT CCCCCAGGAT 3001 CTGGGCTGGG CAACCTCGGT GCCGGGCAGA CGCCAAGGCA CCTGAAACGG CTGCAGTCCC 3061 TGATCCCTTC TGCGCTGGGC TCCGCCTCGC CCGCCAGCAG CCCGTCCCAG GTGGACACAC 3121 CGTCTTCATC CTCCTTCCAC ATCCAACAGC TGGCTGGATT CTCTGCCCCC GCTGGACTGA 3181 GCCCACTCCT GCCCTCATCC AGCTCCTCCC CACCCCCCGG GGCCTGTGGC TCCCCCTCGG 3241 CTCCCACACC ATCAGCTGGC GTAGCCGCCA CCACCATAGC CGGGTTTGGC CACTTCCACA 3301 AGGCGCTGGG TGGCTCCCTG TCCTCCTCCG ACTCTCCCCT GCTCACCCCG CTGCAGCCAG 3361 GCGCCCGCTC CCCGCAGGCT GCCCAGCCAT CTCCCGCGCC ACCCGGGGCC CGGGGAGGCC 3421 TGGGACTCCC GGAGCACTTC CTGCCACCCC CACCCTCATC CAGATCCCCG TCATCTAGCC 3481 CCGGGCAGCT GGGCCAGCCT CCCGGGGAGT TGTCCCTAGG TCTGGCCACT GGCCCACTGA 3541 GCACGCCAGA GACACCCCCA CGGCAGCCTG AGCCGCCGTC CCTTGTGGCA GGGGCCTCTG 3601 GGGGGGCTTC CCCTGTAGGC TTTACTCCCC GAGGAGGTCT CAGCCCCCCT GGCCACAGCC 3661 CAGGCCCCCC AAGAACCTTC CCGAGTGCCC CGCCCCGGGC CTCTGGCTCC CACGGATCCT 3721 TGCTCCTGCC ACCTGCATCC AGCCCCCCAC CACCCCAGGT CCCCCAGCGC CGGGGCACAC 3781 CCCCGCTCAC CCCCGGCCGC CTCACCCAGG ACCTCAAGCT CATCTCCGCG TCTCAGCCAG 3841 CCCTGCCTCA GGACGGGGCG CAGACTCTCC GCAGAGCCTC CCCGCACTCC TCAGGGGAGT 3901 CCATGGCTGC CTTCCCGCTC TTCCCCAGGG CTGGGGGTGG CAGCGGGGGC AGTGGGAGCA 3961 GCGGGGGCCT CGGTCCCCCT GGGAGGCCCT ATGGTGCCAT CCCCGGCCAG CACGTCACTC 4021 TGCCTCGGAA GACATCCTCA GGTTCTTTGC CACCCCCTCT GTCTTTGTTT GGGGCAAGAG 4081 CCACCTCTTC TGGGGGGCCC CCTCTGACTG CTGGACCCCA GAGGGAACCT GGGGCCAGGC 4141 CTGAGCCAGT GCGCTCCAAA CTGCCATCCA ATCTATGAGC TGGGCCCTTC CTTCCCTCTT 4201 CTTTCTTCTT TTCTCTCCCT TCCTTCTTCC TTCAGGTTTA ACTGTGATTA GGAGATATAC 4261 CAATAACAGT AATAATTATT TAAAAAACCA CACACACCAG AAAAACAAAA GACAGCAGAA 4321 AATAACCAGG TATTCTTAGA GCTATAGATT TTTGGTCACT TGCTTTTATA GACTATTTTA 4381 ATACTCAGCA CTAGAGGGAG GGAGGGGGAG GGAGGAGGGA GCAGGCAGGT CCCAAATGCA 4441 AAAGCCAGAG AAAGGCAGAT GGGGTCTCCG GGGCTGGGCA GGGGTGGGAG TGGCCAGTGT 4501 TGGCGGTTCT TAGAGCAGAT GTGTCATTGT GTTCATTTAG AGAAACAGCT GCCATCAGCC 4561 CGTTAGCTGT AACTTGGAGC TCCACTCTGC CCCCAGAAAG GGGCTGCCCT GGGGTGTGCC 4621 CTGGGGAGCC TCAGAAGCCT GCGACCTTGG GAGAAAAGGG CCAGGGCCCT GAGGGCCTAG 4681 CATTTTTTCT ACTGTAAACG TAGCAAGATC TGTATATGAA TATGTATATG TATATGTATG 4741 TAAGATGTGT ATATGTATAG CTATGTAGCG CTCTGTAGAG CCATGTAGAT AGCCACTCAC 4801 ATGTGCGCAC ACGTGTGCGG TCTAGTTTAA TCCCATGTTG ACAGGATGCC CAGGTCACCT 4861 TACACCCAGC AACCCGCCTT GGCCCGCAGG CTGTGCACTG CATGGTCTAG GGACGTTCTC 4921 TCTCCAGTCC TCAGGGAAGA GGACGCCAGG ACTTCGCAGC AGGCCCCCTC TCTCCCCATC 4981 TCTGGTCTCA AAGCCAGTCC CAGCCTGACC TCTCACCACA CGGAAGTGGA AGACTCCCCT 5041 TTCCTAGGGC CTCAAGCACA CACCG

TABLE 5 SEQ ID NO.5 Protein sequence of muHCN2 Accession number: CAA12406 1 MDARGGGGRP GDSPGTTPAP GPPPPPPPPA PPQPQPPPAP PPNPTTPSHP ESADEPGPRA 61 RLCSRDSACT PGAAKGGANG ECGRGEPQCS PEGPARGPKV SFSCRGAASG PSAAEEAGSE 121 EAGPAGEPRG SQASFLQRQF GALLQPGVNK FSLRMFGSQK AVEREQERVK SAGAWIIHPY 181 SDFRFYWDFT MLLFMVGNLI IIPVGITFFK DETTAPWIVF NVVSDTFFLM DLVLNFRTGI 241 VIEDNTEIIL DPEKIKKKYL RTWFVVDFVS SIPVDYIFLI VEKGIDSEVY KTARALRIVR 301 FTKILSLLRL LRLSRLIRYI HQWEEIFHMT YDLASAVMRI CNLISMMLLL CHWDGCLQFL 361 VPMLQDFPSD CWVSINNMVN HSWSELYSFA LFKAMSHMLC IGYGRQAPES MTDIWLTMLS 421 MIVGATCYAM FIGHATALIQ SLDSSRRQYQ EKYKQVEQYM SFHKLPADFR QKIHDYYEHR 481 YQGKMFDEDS ILGELNGPLR EEIVNFNCRK LVASMPLFAN ADPNFVTAML TKLKFEVFQP 541 GDYIIREGTI GKKMYFIQHG VVSVLTKGNK EMKLSDGSYF GEICLLTRGR RTASVRADTY 601 CRLYSLSVDN FNEVLEEYPM MRRAFETVAI DRLDRIGKKN SILLHKVQHD LSSGVFNNQE 661 NAIIQEIVKY DREMVQQAEL GQRVGLFPPP PPPQVTSAIA TLQQAVAMSF CPQVARPLVG 721 PLALGSPRLV RRAPPGPLPP AASPGPPAAS PPAAPSSPRA PRTSPYGVPG SPATRVGPAL 781 PARRLSRASR PLSASQPSLP HGVPAPSPAA SARPASSSTP RLGPAPTART AAPSPDRRDS 841 ASPGAASGLD PLDSARSRLS SNL

TABLE 6 SEQ ID NO. 6 Nucleotide sequence of muHCN2 Accession number: MMJ225122 1 CCGCTCCGCT CCGCACTGCC CGGCGCCGCC TCGCCATGGA TGCGCGCGGG GGCGGCGGGC 61 GGCCGGGCGA TAGTCCGGGC ACGACCCCTG CGCCGGGGCC GCCGCCACCG CCGCCGCCGC 121 CCGCGCCCCC TCAGCCTCAG CCACCACCCG CGCCACCCCC GAACCCCACG ACCCCCTCGC 181 ACCCGGAGTC GGCGGACGAG CCCGGCCCGC GCGCCCGGCT CTGCAGCCGC GACAGCGCCT 241 GCACCCCTGG CGCGGCCAAG GGCGGCGCGA ATGGCGAGTG CGGGCGCGGG GAGCCGCAGT 301 GCAGCCCCGA GGGCCCCGCG CGCGGCCCCA AGGTTTCGTT CTCATGCCGC GGGGCGGCCT 361 CCGGGCCCTC GGCGGCCGAG GAGGCGGGCA GCGAGGAGGC GGGCCCGGCG GGTGAGCCGC 421 GCGGCAGCCA GGCTAGCTTC CTGCAGCGCC AATTCGGGGC GCTTCTGCAG CCCGGCGTCA 481 ACAAGTTCTC CCTGCGGATC TTCGGCAGCC AGAAGGCCGT GGAGCGCGAG CAGGAACGCG 541 TGAAGTCGGC GGGGGCCTGG ATCATCCACC CCTACAGCGA CTTCAGGTTC TACTGGGACT 601 TCACCATGCT GTTGTTCATG GTGGGAAATC TCATTATCAT TCCCGTGGGC ATCACTTTCT 661 TCAAGGACGA GACCACCGCG CCCTGGATCG TCTTCAACGT GGTCTCGGAC ACTTTCTTCC 721 TCATGGACTT GGTGTTGAAC TTCCGCACCG GCATTGTTAT TGAGGACAAC ACGGAGATCA 781 TCCTGGACCC CGAGAAGATA AAGAAGAAGT ACTTGCGTAC GTGGTTCGTG GTGGACTTCG 841 TGTCATCCAT CCCGGTGGAC TACATCTTCC TCATAGTGGA GAAGGGAATC GACTCCGAGG 901 TCTACAAGAC AGCGCGTGCT CTGCGCATCG TGCGCTTCAC CAAGATCCTC AGTCTGCTGC 961 GGCTGCTGCG GCTATCACGG CTCATCCGAT ATATCCACCA GTGGGAAGAG ATTTTCCACA 1021 TGACCTACGA CCTGGCAAGT GCAGTGATGC GCATCTGTAA CCTGATCAGC ATGATGCTAC 1081 TGCTCTGCCA CTGGGACGGT TGCCTGCAGT TCCTGGTGCC CATGCTGCAA GACTTCCCCA 1141 GCGACTGCTG GGTGTCCATC AACAACATGG TGAACCACTC GTGGAGCGAG CTCTACTCGT 1201 TCGCGCTCTT CAAGGCCATG AGCCACATGC TGTGCATCGG CTACGGGCGG CAGGCGCCCG 1261 AGAGCATGAC AGACATCTGG CTGACCATGC TCAGCATGAT CGTAGGCGCC ACCTGCTATG 1321 CCATGTTCAT TGGGCACGCC ACTGCGCTCA TCCAGTCCCT GGATTCGTCA CGGCGCCAAT 1381 ACCAGGAGAA GTACAAGCAA GTAGAGCAAT ACATGTCCTT CCACAAACTG CCCGCTGACT 1441 TCCGCCAGAA GATCCACGAT TACTATGAAC ACCGGTACCA AGGGAAGATG TTTGATGAGG 1501 ACAGCATCCT TGGGGAACTC AACGGGCCAC TGCGTGAGGA GATTGTGAAC TTCAACTGCC 1561 GGAAGCTGGT GGCTTCCATG CCGCTGTTTG CCAATGCAGA CCCCAACTTC GTCACAGCCA 1621 TGCTGACAAA GCTCAAATTT GAGGTCTTCC AGCCTGGAGA TTACATCATC CGAGAGGGGA 1681 CCATCGGGAA GAAGATGTAC TTCATCCAGC ATGGGGTGGT GAGCGTGCTC ACCAAGGGCA 1741 ACAAGGAGAT GAAGCTGTCG GATGGCTCCT ATTTCGGGGA GATCTGCTTG CTCACGAGGG 1801 GCCGGCGTAC GGCCAGCGTG CGAGCTGACA CCTACTGTCG CCTCTACTCA CTGAGTGTGG 1861 ACAATTTCAA CGAGGTGCTG GAGGAATACC CCATGATGCG GCGTGCCTTT GAGACTGTGG 1921 CTATTGACCG GCTAGATCGC ATAGGCAAGA AGAACTCCAT CTTGCTGCAC AAGGTTCAGC 1981 ATGATCTCAG CTCAGGTGTG TTCAACAACC AGGAGAATGC CATCATCCAG GAGATTGTCA 2041 AATATGACCG TGAGATGGTG CAGCAGGCAG AGCTTGGCCA GCGTGTGGGG CTCTTCCCAC 2101 CACCGCCACC ACCGCAGGTC ACATCGGCCA TTGCCACCCT ACAGCAGGCT GTGGCCATGA 2161 GCTTCTGCCC GCAGGTGGCC CGCCCGCTCG TGGGGCCCCT GGCGCTAGGC TCCCCACGCC 2221 TAGTGCGCCG CGCGCCCCCA GGGCCTCTGC CTCCTGCAGC CTCGCCAGGG CCACCCGCAG 2281 CAAGCCCCCC GGCTGCACCC TCGAGCCCTC GGGCACCGCG GACCTCACCC TACGGTGTGC 2341 CTGGCTCTCC GGCAACGCGC GTGGGGCCCG CATTGCCCGC ACGTCGCCTG AGCCGCGCCT 2401 CGCGCCCACT GTCCGCCTCG CAGCCCTCGC TGCCCCATGG CGTGCCCGCG CCCAGCCCCG 2461 CGGCCTCTGC GCGCCCGGCC AGCAGCTCCA CGCCGCGCCT GGGACCCGCA CCCACCGCCC 2521 GGACCGCCGC GCCCAGTCCG GACCGCAGGG ACTCAGCCTC GCCGGGCGCT GCCAGTGGCC 2581 TCGACCCACT GGACTCTGCG CGCTCGCGCC TCTCTTCCAA CTTGTGACCC TTGAGCGCCG 2641 CCCCGCGGGC CGGGCGGGGC CGTCATCCAC ACCAAAGCCA TGCCTCGCGC CGCCCGCCCG 2701 TGCCCGTGCA GAAGCCATAG AGGGACGTAG GTAGCTTAGG AGGCGGGCGG CCCTGCGCCC 2761 GGCTGTCCCC CCATCGCCCT GCGCCCACCC CCATCGCCCC TGCCCCAGCG GCGGCCGCAC 2821 GGGAGAGGGA GGGGTGCGAT CACCTCGGTG CCTCAGCCCC AACCTGGGAC AGGGACAGGG 2881 CGGCCCTGGC CGAGGACCTG GCTGTGCCCC GCATGTGCGG TGGCCTCCGA GGAAGAATAT 2941 GGATCAAGTG CAATACACGG CCAAGCCGGC GTGGGGGTGA GGCTGGGTCC CCGGCCGTCG 3001 CCATGAATGT ACTGACGAGC CGAGGCAGCA GTGGCCCCCA CGCCCCATTA ACCCACAACC 3061 CCATTCCGCG CAATAAACGA CAGCATTGGC AAAAAAAAAA AA //

TABLE 7 Abbreviations AKT Arabidopsis thaliana K+ transport cAMP cyclic adenosine monophosphate CHO Chinese hamster ovary EDTA ethylenediamine tetraacetic acid FLIPR fluorescence imaging plate reader HAC hyperpolarization-activated cation channel; this name was used by some groups HCN hyperpolarization-activated cyclic nucleotide gated cation channel; this is the new, generally accepted term HEK human embryonic kidney; HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid HTS high-thoughput screening KAT K+ channel from Arabidopsis thaliana

SEQUENCE LISTING <100> GENERAL INFORMATION: <160> NUMBER OF SEQ ID NOS: 10 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 1 <211> LENGTH: 889 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 1 Met Asp Ala Arg Gly Gly Gly Gly Arg Pro Gly Glu Ser Pro Gly Ala 1 5 10 15 Ser Pro Thr Thr Gly Pro Pro Pro Pro Pro Pro Pro Arg Pro Pro Lys 20 25 30 Gln Gln Pro Pro Pro Pro Pro Pro Pro Ala Pro Pro Pro Gly Pro Gly 35 40 45 Pro Ala Pro Pro Gln His Pro Pro Arg Ala Glu Ala Leu Pro Pro Glu 50 55 60 Ala Ala Asp Glu Gly Gly Pro Arg Gly Arg Leu Arg Ser Arg Asp Ser 65 70 75 80 Ser Cys Gly Arg Pro Gly Thr Pro Gly Ala Ala Ser Thr Ala Lys Gly 85 90 95 Ser Pro Asn Gly Glu Cys Gly Arg Gly Glu Pro Gln Cys Ser Pro Ala 100 105 110 Gly Pro Glu Gly Pro Ala Arg Gly Pro Lys Val Ser Phe Ser Cys Arg 115 120 125 Gly Ala Ala Ser Gly Pro Ala Pro Gly Pro Gly Pro Ala Glu Glu Ala 130 135 140 Gly Ser Glu Glu Ala Gly Pro Ala Gly Glu Pro Arg Gly Ser Gln Ala 145 150 155 160 Ser Phe Met Gln Arg Gln Phe Gly Ala Leu Leu Gln Pro Gly Val Asn 165 170 175 Lys Phe Ser Leu Arg Met Phe Gly Ser Gln Lys Ala Val Glu Arg Glu 180 185 190 Gln Glu Arg Val Lys Ser Ala Gly Ala Trp Ile Ile His Pro Tyr Ser 195 200 205 Asp Phe Arg Phe Tyr Trp Asp Phe Thr Met Leu Leu Phe Met Val Gly 210 215 220 Asn Leu Ile Ile Ile Pro Val Gly Ile Thr Phe Phe Lys Asp Glu Thr 225 230 235 240 Thr Ala Pro Trp Ile Val Phe Asn Val Val Ser Asp Thr Phe Phe Leu 245 250 255 Met Asp Leu Val Leu Asn Phe Arg Thr Gly Ile Val Ile Glu Asp Asn 260 265 270 Thr Glu Ile Ile Leu Asp Pro Glu Lys Ile Lys Lys Lys Tyr Leu Arg 275 280 285 Thr Trp Phe Val Val Asp Phe Val Ser Ser Ile Pro Val Asp Tyr Ile 290 295 300 Phe Leu Ile Val Glu Lys Gly Ile Asp Ser Glu Val Tyr Lys Thr Ala 305 310 315 320 Arg Ala Leu Arg Ile Val Arg Phe Thr Lys Ile Leu Ser Leu Leu Arg 325 330 335 Leu Leu Arg Leu Ser Arg Leu Ile Arg Tyr Ile His Gln Trp Glu Glu 340 345 350 Ile Phe His Met Thr Tyr Asp Leu Ala Ser Ala Val Met Arg Ile Cys 355 360 365 Asn Leu Ile Ser Met Met Leu Leu Leu Cys His Trp Asp Gly Cys Leu 370 375 380 Gln Phe Leu Val Pro Met Leu Gln Asp Phe Pro Arg Asn Cys Trp Val 385 390 395 400 Ser Ile Asn Gly Met Val Asn His Ser Trp Ser Glu Leu Tyr Ser Phe 405 410 415 Ala Leu Phe Lys Ala Met Ser His Met Leu Cys Ile Gly Tyr Gly Arg 420 425 430 Gln Ala Pro Glu Ser Met Thr Asp Ile Trp Leu Thr Met Leu Ser Met 435 440 445 Ile Val Gly Ala Thr Cys Tyr Ala Met Phe Ile Gly His Ala Thr Ala 450 455 460 Leu Ile Gln Ser Leu Asp Ser Ser Arg Arg Gln Tyr Gln Glu Lys Tyr 465 470 475 480 Lys Gln Val Glu Gln Tyr Met Ser Phe His Lys Leu Pro Ala Asp Phe 485 490 495 Arg Gln Lys Ile His Asp Tyr Tyr Glu His Arg Tyr Gln Gly Lys Met 500 505 510 Phe Asp Glu Asp Ser Ile Leu Gly Glu Leu Asn Gly Pro Leu Arg Glu 515 520 525 Glu Ile Val Asn Phe Asn Cys Arg Lys Leu Val Ala Ser Met Pro Leu 530 535 540 Phe Ala Asn Ala Asp Pro Asn Phe Val Thr Ala Met Leu Thr Lys Leu 545 550 555 560 Lys Phe Glu Val Phe Gln Pro Gly Asp Tyr Ile Ile Arg Glu Gly Thr 565 570 575 Ile Gly Lys Lys Met Tyr Phe Ile Gln His Gly Val Val Ser Val Leu 580 585 590 Thr Lys Gly Asn Lys Glu Met Lys Leu Ser Asp Gly Ser Tyr Phe Gly 595 600 605 Glu Ile Cys Leu Leu Thr Arg Gly Arg Arg Thr Ala Ser Val Arg Ala 610 615 620 Asp Thr Tyr Cys Arg Leu Tyr Ser Leu Ser Val Asp Asn Phe Asn Glu 625 630 635 640 Val Leu Glu Glu Tyr Pro Met Met Arg Arg Ala Phe Glu Thr Val Ala 645 650 655 Ile Asp Arg Leu Asp Arg Ile Gly Lys Lys Asn Ser Ile Leu Leu His 660 665 670 Lys Val Gln His Asp Leu Asn Ser Gly Val Phe Asn Asn Gln Glu Asn 675 680 685 Ala Ile Ile Gln Glu Ile Val Lys Tyr Asp Arg Glu Met Val Gln Gln 690 695 700 Ala Glu Leu Gly Gln Arg Val Gly Leu Phe Pro Pro Pro Pro Pro Pro 705 710 715 720 Pro Gln Val Thr Ser Ala Ile Ala Thr Leu Gln Gln Ala Ala Ala Met 725 730 735 Ser Phe Cys Pro Gln Val Ala Arg Pro Leu Val Gly Pro Leu Ala Leu 740 745 750 Gly Ser Pro Arg Leu Val Arg Arg Pro Pro Pro Gly Pro Ala Pro Ala 755 760 765 Ala Ala Ser Pro Gly Pro Pro Pro Pro Ala Ser Pro Pro Gly Ala Pro 770 775 780 Ala Ser Pro Arg Ala Pro Arg Thr Ser Pro Tyr Gly Gly Leu Pro Ala 785 790 795 800 Ala Pro Leu Ala Gly Pro Ala Leu Pro Ala Arg Arg Leu Ser Arg Ala 805 810 815 Ser Arg Pro Leu Ser Ala Ser Gln Pro Ser Leu Pro His Gly Ala Pro 820 825 830 Gly Pro Ala Ala Ser Thr Arg Pro Ala Ser Ser Ser Thr Pro Arg Leu 835 840 845 Gly Pro Thr Pro Ala Ala Arg Ala Ala Ala Pro Ser Pro Asp Arg Arg 850 855 860 Asp Ser Ala Ser Pro Gly Ala Ala Gly Gly Leu Asp Pro Gln Asp Ser 865 870 875 880 Ala Arg Ser Arg Leu Ser Ser Asn Leu 885 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 2 <211> LENGTH: 3372 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 2 cggctccgct ccgcactgcc cggcgccgcc tcgccatgga cgcgcgcggg ggcggcgggc 60 ggcccgggga gagcccgggc gcgagcccca cgaccgggcc gccgccgccg ccgcccccgc 120 gcccccccaa acagcagccg ccgccgccgc cgccgcccgc gccccccccg ggccccgggc 180 ccgcgccccc ccagcacccg ccccgggccg aggcgttgcc cccggaggcg gcggatgagg 240 gcggcccgcg gggccggctc cgcagccgcg acagctcgtg cggccgcccc ggcaccccgg 300 gcgcggcgag cacggccaag ggcagcccga acggcgagtg cgggcgcggc gagccgcagt 360 gcagccccgc ggggcccgag ggcccggcgc gggggcccaa ggtgtcgttc tcgtgccgcg 420 gggcggcctc ggggcccgcg ccggggccgg ggccggcgga ggaggcgggc agcgaggagg 480 cgggcccggc gggggagccg cgcggcagcc aggccagctt catgcagcgc cagttcggcg 540 cgctcctgca gccgggcgtc aacaagttct cgctgcggat gttcggcagc cagaaggccg 600 tggagcgcga gcaggagcgc gtcaagtcgg cgggggcctg gatcatccac ccgtacagcg 660 acttcaggtt ctactgggac ttcaccatgc tgctgttcat ggtgggaaac ctcatcatca 720 tcccagtggg catcaccttc ttcaaggatg agaccactgc cccgtggatc gtgttcaacg 780 tggtctcgga caccttcttc ctcatggacc tggtgttgaa cttccgcacc ggcattgtga 840 tcgaggacaa cacggagatc atcctggacc ccgagaagat caagaagaag tatctgcgca 900 cgtggttcgt ggtggacttc gtgtcctcca tccccgtgga ctacatcttc cttatcgtgg 960 agaagggcat tgactccgag gtctacaaga cggcacgcgc cctgcgcatc gtgcgcttca 1020 ccaagatcct cagcctcctg cggctgctgc gcctctcacg cctgatccgc tacatccatc 1080 agtgggagga gatcttccac atgacctatg acctggccag cgcggtgatg aggatctgca 1140 atctcatcag catgatgctg ctgctctgcc actgggacgg ctgcctgcag ttcctggtgc 1200 ctatgctgca ggacttcccg cgcaactgct gggtgtccat caatggcatg gtgaaccact 1260 cgtggagtga actgtactcc ttcgcactct tcaaggccat gagccacatg ctgtgcatcg 1320 ggtacggccg gcaggcgccc gagagcatga cggacatctg gctgaccatg ctcagcatga 1380 ttgtgggtgc cacctgctac gccatgttca tcggccacgc cactgccctc atccagtcgc 1440 tggactcctc gcggcgccag taccaggaga agtacaagca ggtggagcag tacatgtcct 1500 tccacaagct gccagctgac ttccgccaga agatccacga ctactatgag caccgttacc 1560 agggcaagat gtttgacgag gacagcatcc tgggcgagct caacgggccc ctgcgggagg 1620 agatcgtcaa cttcaactgc cggaagctgg tggcctccat gccgctgttc gccaacgccg 1680 accccaactt cgtcacggcc atgctgacca agctcaagtt cgaggtcttc cagccgggtg 1740 actacatcat ccgcgaaggc accatcggga agaagatgta cttcatccag cacggcgtgg 1800 tcagcgtgct cactaagggc aacaaggaga tgaagctgtc cgatggctcc tacttcgggg 1860 agatctgcct gctcacccgg ggccgccgca cggcgagcgt gcgggctgac acctactgcc 1920 gcctctattc gctgagcgtg gacaacttca acgaggtgct ggaggagtac cccatgatgc 1980 ggcgcgcctt cgagacggtg gccatcgacc gcctggaccg catcggcaag aagaattcca 2040 tcctcctgca caaggtgcag catgacctca actcgggcgt attcaacaac caggagaacg 2100 ccatcatcca ggagatcgtc aagtacgacc gcgagatggt gcagcaggcc gagctgggtc 2160 agcgcgtggg cctcttcccg ccgccgccgc cgccgccgca ggtcacctcg gccatcgcca 2220 cgctgcagca ggcggcggcc atgagcttct gcccgcaggt ggcgcggccg ctcgtggggc 2280 cgctggcgct cggctcgccg cgcctcgtgc gccgcccgcc cccggggccc gcacctgccg 2340 ccgcctcacc cgggcccccg ccccccgcca gccccccggg cgcgcccgcc agcccccggg 2400 caccgcggac ctcgccctac ggcggcctgc ccgccgcccc ccttgctggg cccgccctgc 2460 ccgcgcgccg cctgagccgc gcgtcgcgcc cactgtccgc ctcgcagccc tcgctgcctc 2520 acggcgcccc cggccccgcg gcctccacac gcccggccag cagctccaca ccgcgcttgg 2580 ggcccacgcc cgctgcccgg gccgccgcgc ccagcccgga ccgcagggac tcggcctcac 2640 ccggcgccgc cggcggcctg gacccccagg actccgcgcg ctcgcgcctc tcgtccaact 2700 tgtgaccctc gccgaccgcc ccgcgggccc aggcgggccg ggggcggggc cgtcatccag 2760 accaaagcca tgccattgcg ctgccccggc cgccagtccg cccagaagcc atagacgaga 2820 cgtaggtagc cgtagttgga cggacgggca gggccggcgg ggcagccccc tccgcgcccc 2880 cggccgtccc ccctcatcgc cccgcgccca cccccatcgc ccctgccccc ggcggcggcc 2940 tcgcgtgcga gggggctccc ttcacctcgg tgcctcagtt cccccagctg taagacaggg 3000 acggggcggc ccagtggctg agaggagccg gctgtggagc cccgcccgcc ccccaccctc 3060 taggtggccc ccgtccgagg aggatcgttt tctaagtgca atacttggcc cgccggcttc 3120 ccgctgcccc catcgcgctc acgcaataac cggcccggcc cccgtccgcg cgcgtccccc 3180 ggtgacctcg gggagcagca ccccgcctcc ctccagcact ggcaccgaga ggcaggcctg 3240 gctgcgcagg gcgcgggggg gaggctgggg tcccgccgcc gtgatgaatg tactgacgag 3300 ccgaggcagc agtgccccca ccgtggcccc ccacgcccca ttaaccccca cacccccatt 3360 ccgcgcaata aa 3372 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 3 <211> LENGTH: 1203 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 3 Met Asp Lys Leu Pro Pro Ser Met Arg Lys Arg Leu Tyr Ser Leu Pro 1 5 10 15 Gln Gln Val Gly Ala Lys Ala Trp Ile Met Asp Glu Glu Glu Asp Ala 20 25 30 Glu Glu Glu Gly Ala Gly Gly Arg Gln Asp Pro Ser Arg Arg Ser Ile 35 40 45 Arg Leu Arg Pro Leu Pro Ser Pro Ser Pro Ser Ala Ala Ala Gly Gly 50 55 60 Thr Glu Ser Arg Ser Ser Ala Leu Gly Ala Ala Asp Ser Glu Gly Pro 65 70 75 80 Ala Arg Gly Ala Gly Lys Ser Ser Thr Asn Gly Asp Cys Arg Arg Phe 85 90 95 Arg Gly Ser Leu Ala Ser Leu Gly Ser Arg Gly Gly Gly Ser Gly Gly 100 105 110 Thr Gly Ser Gly Ser Ser His Gly His Leu His Asp Ser Ala Glu Glu 115 120 125 Arg Arg Leu Ile Ala Glu Gly Asp Ala Ser Pro Gly Glu Asp Arg Thr 130 135 140 Pro Pro Gly Leu Ala Ala Glu Pro Glu Arg Pro Gly Ala Ser Ala Gln 145 150 155 160 Pro Ala Ala Ser Pro Pro Pro Pro Gln Gln Pro Pro Gln Pro Ala Ser 165 170 175 Ala Ser Cys Glu Gln Pro Ser Val Asp Thr Ala Ile Lys Val Glu Gly 180 185 190 Gly Ala Ala Ala Gly Asp Gln Ile Leu Pro Glu Ala Glu Val Arg Leu 195 200 205 Gly Gln Ala Gly Phe Met Gln Arg Gln Phe Gly Ala Met Leu Gln Pro 210 215 220 Gly Val Asn Lys Phe Ser Leu Arg Met Phe Gly Ser Gln Lys Ala Val 225 230 235 240 Glu Arg Glu Gln Glu Arg Val Lys Ser Ala Gly Phe Trp Ile Ile His 245 250 255 Pro Tyr Ser Asp Phe Arg Phe Tyr Trp Asp Leu Thr Met Leu Leu Leu 260 265 270 Met Val Gly Asn Leu Ile Ile Ile Pro Val Gly Ile Thr Phe Phe Lys 275 280 285 Asp Glu Asn Thr Thr Pro Trp Ile Val Phe Asn Val Val Ser Asp Thr 290 295 300 Phe Phe Leu Ile Asp Leu Val Leu Asn Phe Arg Thr Gly Ile Val Val 305 310 315 320 Glu Asp Asn Thr Glu Ile Ile Leu Asp Pro Gln Arg Ile Lys Met Lys 325 330 335 Tyr Leu Lys Ser Trp Phe Met Val Asp Phe Ile Ser Ser Ile Pro Val 340 345 350 Asp Tyr Ile Phe Leu Ile Val Glu Thr Arg Ile Asp Ser Glu Val Tyr 355 360 365 Lys Thr Ala Arg Ala Leu Arg Ile Val Arg Phe Thr Lys Ile Leu Ser 370 375 380 Leu Leu Arg Leu Leu Arg Leu Ser Arg Leu Ile Arg Tyr Ile His Gln 385 390 395 400 Trp Glu Glu Ile Phe His Met Thr Tyr Asp Leu Ala Ser Ala Val Val 405 410 415 Arg Ile Val Asn Leu Ile Gly Met Met Leu Leu Leu Cys His Trp Asp 420 425 430 Gly Cys Leu Gln Phe Leu Val Pro Met Leu Gln Asp Phe Pro Asp Asp 435 440 445 Cys Trp Val Ser Ile Asn Asn Met Val Asn Asn Ser Trp Gly Lys Gln 450 455 460 Tyr Ser Tyr Ala Leu Phe Lys Ala Met Ser His Met Leu Cys Ile Gly 465 470 475 480 Tyr Gly Arg Gln Ala Pro Val Gly Met Ser Asp Val Trp Leu Thr Met

485 490 495 Leu Ser Met Ile Val Gly Ala Thr Cys Tyr Ala Met Phe Ile Gly His 500 505 510 Ala Thr Ala Leu Ile Gln Ser Leu Asp Ser Ser Arg Arg Gln Tyr Gln 515 520 525 Glu Lys Tyr Lys Gln Val Glu Gln Tyr Met Ser Phe His Lys Leu Pro 530 535 540 Pro Asp Thr Arg Gln Arg Ile His Asp Tyr Tyr Glu His Arg Tyr Gln 545 550 555 560 Gly Lys Met Phe Asp Glu Glu Ser Ile Leu Gly Glu Leu Ser Glu Pro 565 570 575 Leu Arg Glu Glu Ile Ile Asn Phe Asn Cys Arg Lys Leu Val Ala Ser 580 585 590 Met Pro Leu Phe Ala Asn Ala Asp Pro Asn Phe Val Thr Ser Met Leu 595 600 605 Thr Lys Leu Arg Phe Glu Val Phe Gln Pro Gly Asp Tyr Ile Ile Arg 610 615 620 Glu Gly Thr Ile Gly Lys Lys Met Tyr Phe Ile Gln His Gly Val Val 625 630 635 640 Ser Val Leu Thr Lys Gly Asn Lys Glu Thr Lys Leu Ala Asp Gly Ser 645 650 655 Tyr Phe Gly Glu Ile Cys Leu Leu Thr Arg Gly Arg Arg Thr Ala Ser 660 665 670 Val Arg Ala Asp Thr Tyr Cys Arg Leu Tyr Ser Leu Ser Val Asp Asn 675 680 685 Phe Asn Glu Val Leu Glu Glu Tyr Pro Met Met Arg Arg Ala Phe Glu 690 695 700 Thr Val Ala Leu Asp Arg Leu Asp Arg Ile Gly Lys Lys Asn Ser Ile 705 710 715 720 Leu Leu His Lys Val Gln His Asp Leu Asn Ser Gly Val Phe Asn Tyr 725 730 735 Gln Glu Asn Glu Ile Ile Gln Gln Ile Val Gln His Asp Arg Glu Met 740 745 750 Ala His Cys Ala His Arg Val Gln Ala Ala Ala Ser Ala Thr Pro Thr 755 760 765 Pro Thr Pro Val Ile Trp Thr Pro Leu Ile Gln Ala Pro Leu Gln Ala 770 775 780 Ala Ala Ala Thr Thr Ser Val Ala Ile Ala Leu Thr His His Pro Arg 785 790 795 800 Leu Pro Ala Ala Ile Phe Arg Pro Pro Pro Gly Ser Gly Leu Gly Asn 805 810 815 Leu Gly Ala Gly Gln Thr Pro Arg His Leu Lys Arg Leu Gln Ser Leu 820 825 830 Ile Pro Ser Ala Leu Gly Ser Ala Ser Pro Ala Ser Ser Pro Ser Gln 835 840 845 Val Asp Thr Pro Ser Ser Ser Ser Phe His Ile Gln Gln Leu Ala Gly 850 855 860 Phe Ser Ala Pro Ala Gly Leu Ser Pro Leu Leu Pro Ser Ser Ser Ser 865 870 875 880 Ser Pro Pro Pro Gly Ala Cys Gly Ser Pro Ser Ala Pro Thr Pro Ser 885 890 895 Ala Gly Val Ala Ala Thr Thr Ile Ala Gly Phe Gly His Phe His Lys 900 905 910 Ala Leu Gly Gly Ser Leu Ser Ser Ser Asp Ser Pro Leu Leu Thr Pro 915 920 925 Leu Gln Pro Gly Ala Arg Ser Pro Gln Ala Ala Gln Pro Ser Pro Ala 930 935 940 Pro Pro Gly Ala Arg Gly Gly Leu Gly Leu Pro Glu His Phe Leu Pro 945 950 955 960 Pro Pro Pro Ser Ser Arg Ser Pro Ser Ser Ser Pro Gly Gln Leu Gly 965 970 975 Gln Pro Pro Gly Glu Leu Ser Leu Gly Leu Ala Thr Gly Pro Leu Ser 980 985 990 Thr Pro Glu Thr Pro Pro Arg Gln Pro Glu Pro Pro Ser Leu Val Ala 995 1000 1005 Gly Ala Ser Gly Gly Ala Ser Pro Val Gly Phe Thr Pro Arg Gly Gly 1010 1015 1020 Leu Ser Pro Pro Gly His Ser Pro Gly Pro Pro Arg Thr Phe Pro Ser 1025 1030 1035 1040 Ala Pro Pro Arg Ala Ser Gly Ser His Gly Ser Leu Leu Leu Pro Pro 1045 1050 1055 Ala Ser Ser Pro Pro Pro Pro Gln Val Pro Gln Arg Arg Gly Thr Pro 1060 1065 1070 Pro Leu Thr Pro Gly Arg Leu Thr Gln Asp Leu Lys Leu Ile Ser Ala 1075 1080 1085 Ser Gln Pro Ala Leu Pro Gln Asp Gly Ala Gln Thr Leu Arg Arg Ala 1090 1095 1100 Ser Pro His Ser Ser Gly Glu Ser Met Ala Ala Phe Pro Leu Phe Pro 1105 1110 1115 1120 Arg Ala Gly Gly Gly Ser Gly Gly Ser Gly Ser Ser Gly Gly Leu Gly 1125 1130 1135 Pro Pro Gly Arg Pro Tyr Gly Ala Ile Pro Gly Gln His Val Thr Leu 1140 1145 1150 Pro Arg Lys Thr Ser Ser Gly Ser Leu Pro Pro Pro Leu Ser Leu Phe 1155 1160 1165 Gly Ala Arg Ala Thr Ser Ser Gly Gly Pro Pro Leu Thr Ala Gly Pro 1170 1175 1180 Gln Arg Glu Pro Gly Ala Arg Pro Glu Pro Val Arg Ser Lys Leu Pro 1185 1190 1195 1200 Ser Asn Leu <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 4 <211> LENGTH: 5065 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 4 ggtcgctggg ctccgctcgg ttgcggcggg agccccggga cgggccggac gggccggggc 60 agaggaggcg aggcgagctc gcgggtggcc agccacaaag cccgggcggc gagacagacg 120 gacagccagc cctcccgcgg gacgcacgcc cgggacccgc gcgggccgtg cgctctgcac 180 tccggagcgg ttccctgagc gccgcggccg cagagcctct ccggccggcg cccattgttc 240 cccgcggggg cggggcgcct ggagccgggc ggcgcgccgc gcccctgaac gccagaggga 300 gggagggagg caagaaggga gcgcggggtc cccgcgccca gccgggcccg ggaggaggtg 360 tagcgcggcg agcccgggga ctcggagcgg gactaggatc ctccccgcgg cgcgcagcct 420 gcccaagcat gggcgcctga ggctgccccc acgccggcgg caaaggacgc gtccccacgg 480 gcggactgac cggcgggcgg acctggagcc cgtccgcggc gccgcgctcc tgcccccggc 540 ccggtccgac cccggcccct ggcgccatgg acaagctgcc gccgtccatg cgcaagcggc 600 tctacagcct cccgcagcag gtgggggcca aggcgtggat catggacgag gaagaggacg 660 ccgaggagga gggggccggg ggccgccaag accccagccg caggagcatc cggctgcggc 720 cactgccctc gccctccccc tcggcggccg cgggtggcac ggagtcccgg agctcggccc 780 tcggggcagc ggacagcgaa gggccggccc gcggcgcggg caagtccagc acgaacggcg 840 actgcaggcg cttccgcggg agcctggcct cgctgggcag ccggggcggc ggcagcggcg 900 gcacggggag cggcagcagt cacggacacc tgcatgactc cgcggaggag cggcggctca 960 tcgccgaggg cgacgcgtcc cccggcgagg acaggacgcc cccaggcctg gcggccgagc 1020 ccgagcgccc cggcgcctcg gcgcagcccg cagcctcgcc gccgccgccc cagcagccac 1080 cgcagccggc ctccgcctcc tgcgagcagc cctcggtgga caccgctatc aaagtggagg 1140 gaggcgcggc tgccggcgac cagatcctcc cggaggccga ggtgcgcctg ggccaggccg 1200 gcttcatgca gcgccagttc ggggccatgc tccaacccgg ggtcaacaaa ttctccctaa 1260 ggatgttcgg cagccagaaa gccgtggagc gcgaacagga gagggtcaag tcggccggat 1320 tttggattat ccacccctac agtgacttca gattttactg ggacctgacc atgctgctgc 1380 tgatggtggg aaacctgatt atcattcctg tgggcatcac cttcttcaag gatgagaaca 1440 ccacaccctg gattgtcttc aatgtggtgt cagacacatt cttcctcatc gacttggtcc 1500 tcaacttccg cacagggatc gtggtggagg acaacacaga gatcatcctg gacccgcagc 1560 ggattaaaat gaagtacctg aaaagctggt tcatggtaga tttcatttcc tccatccccg 1620 tggactacat cttcctcatt gtggagacac gcatcgactc ggaggtctac aagactgccc 1680 gggccctgcg cattgtccgc ttcacgaaga tcctcagcct cttacgcctg ttacgcctct 1740 cccgcctcat tcgatatatt caccagtggg aagagatctt ccacatgacc tacgacctgg 1800 ccagcgccgt ggtgcgcatc gtgaacctca tcggcatgat gctcctgctc tgccactggg 1860 acggctgcct gcagttcctg gtacccatgc tacaggactt ccctgacgac tgctgggtgt 1920 ccatcaacaa catggtgaac aactcctggg ggaagcagta ctcctacgcg ctcttcaagg 1980 ccatgagcca catgctgtgc atcggctacg ggcggcaggc gcccgtgggc atgtccgacg 2040 tctggctcac catgctcagc atgatcgtgg gtgccacctg ctacgccatg ttcattggcc 2100 acgccactgc cctcatccag tccctggact cctcccggcg ccagtaccag gaaaagtaca 2160 agcaggtgga gcagtacatg tcctttcaca agctcccgcc cgacacccgg cagcgcatcc 2220 acgactacta cgagcaccgc taccagggca agatgttcga cgaggagagc atcctgggcg 2280 agctaagcga gcccctgcgg gaggagatca tcaactttaa ctgtcggaag ctggtggcct 2340 ccatgccact gtttgccaat gcggacccca acttcgtgac gtccatgctg accaagctgc 2400 gtttcgaggt cttccagcct ggggactaca tcatccggga aggcaccatt ggcaagaaga 2460 tgtacttcat ccagcatggc gtggtcagcg tgctcaccaa gggcaacaag gagaccaagc 2520 tggccgacgg ctcctacttt ggagagatct gcctgctgac ccggggccgg cgcacagcca 2580 gcgtgagggc cgacacctac tgccgcctct actcgctgag cgtggacaac ttcaatgagg 2640 tgctggagga gtaccccatg atgcgaaggg ccttcgagac cgtggcgctg gaccgcctgg 2700 accgcattgg caagaagaac tccatcctcc tccacaaagt ccagcacgac ctcaactccg 2760 gcgtcttcaa ctaccaggag aatgagatca tccagcagat tgtgcagcat gaccgggaga 2820 tggcccactg cgcgcaccgc gtccaggctg ctgcctctgc caccccaacc cccacgcccg 2880 tcatctggac cccgctgatc caggcaccac tgcaggctgc cgctgccacc acttctgtgg 2940 ccatagccct cacccaccac cctcgcctgc ctgctgccat cttccgccct cccccaggat 3000 ctgggctggg caacctcggt gccgggcaga cgccaaggca cctgaaacgg ctgcagtccc 3060 tgatcccttc tgcgctgggc tccgcctcgc ccgccagcag cccgtcccag gtggacacac 3120 cgtcttcatc ctccttccac atccaacagc tggctggatt ctctgccccc gctggactga 3180 gcccactcct gccctcatcc agctcctccc caccccccgg ggcctgtggc tccccctcgg 3240 ctcccacacc atcagctggc gtagccgcca ccaccatagc cgggtttggc cacttccaca 3300 aggcgctggg tggctccctg tcctcctccg actctcccct gctcaccccg ctgcagccag 3360 gcgcccgctc cccgcaggct gcccagccat ctcccgcgcc acccggggcc cggggaggcc 3420 tgggactccc ggagcacttc ctgccacccc caccctcatc cagatccccg tcatctagcc 3480 ccgggcagct gggccagcct cccggggagt tgtccctagg tctggccact ggcccactga 3540 gcacgccaga gacaccccca cggcagcctg agccgccgtc ccttgtggca ggggcctctg 3600 ggggggcttc ccctgtaggc tttactcccc gaggaggtct cagcccccct ggccacagcc 3660 caggcccccc aagaaccttc ccgagtgccc cgccccgggc ctctggctcc cacggatcct 3720 tgctcctgcc acctgcatcc agccccccac caccccaggt cccccagcgc cggggcacac 3780 ccccgctcac ccccggccgc ctcacccagg acctcaagct catctccgcg tctcagccag 3840 ccctgcctca ggacggggcg cagactctcc gcagagcctc cccgcactcc tcaggggagt 3900 ccatggctgc cttcccgctc ttccccaggg ctgggggtgg cagcgggggc agtgggagca 3960 gcgggggcct cggtccccct gggaggccct atggtgccat ccccggccag cacgtcactc 4020 tgcctcggaa gacatcctca ggttctttgc caccccctct gtctttgttt ggggcaagag 4080 ccacctcttc tggggggccc cctctgactg ctggacccca gagggaacct ggggccaggc 4140 ctgagccagt gcgctccaaa ctgccatcca atctatgagc tgggcccttc cttccctctt 4200 ctttcttctt ttctctccct tccttcttcc ttcaggttta actgtgatta ggagatatac 4260 caataacagt aataattatt taaaaaacca cacacaccag aaaaacaaaa gacagcagaa 4320 aataaccagg tattcttaga gctatagatt tttggtcact tgcttttata gactatttta 4380 atactcagca ctagagggag ggagggggag ggaggaggga gcaggcaggt cccaaatgca 4440 aaagccagag aaaggcagat ggggtctccg gggctgggca ggggtgggag tggccagtgt 4500 tggcggttct tagagcagat gtgtcattgt gttcatttag agaaacagct gccatcagcc 4560 cgttagctgt aacttggagc tccactctgc ccccagaaag gggctgccct ggggtgtgcc 4620 ctggggagcc tcagaagcct gcgaccttgg gagaaaaggg ccagggccct gagggcctag 4680 cattttttct actgtaaacg tagcaagatc tgtatatgaa tatgtatatg tatatgtatg 4740 taagatgtgt atatgtatag ctatgtagcg ctctgtagag ccatgtagat agccactcac 4800 atgtgcgcac acgtgtgcgg tctagtttaa tcccatgttg acaggatgcc caggtcacct 4860 tacacccagc aacccgcctt ggcccgcagg ctgtgcactg catggtctag ggacgttctc 4920 tctccagtcc tcagggaaga ggacgccagg acttcgcagc aggccccctc tctccccatc 4980 tctggtctca aagccagtcc cagcctgacc tctcaccaca cggaagtgga agactcccct 5040 ttcctagggc ctcaagcaca caccg 5065 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 5 <211> LENGTH: 863 <212> TYPE: PRT <213> ORGANISM: Murinae gen. sp. <400> SEQUENCE: 5 Met Asp Ala Arg Gly Gly Gly Gly Arg Pro Gly Asp Ser Pro Gly Thr 1 5 10 15 Thr Pro Ala Pro Gly Pro Pro Pro Pro Pro Pro Pro Pro Ala Pro Pro 20 25 30 Gln Pro Gln Pro Pro Pro Ala Pro Pro Pro Asn Pro Thr Thr Pro Ser 35 40 45 His Pro Glu Ser Ala Asp Glu Pro Gly Pro Arg Ala Arg Leu Cys Ser 50 55 60 Arg Asp Ser Ala Cys Thr Pro Gly Ala Ala Lys Gly Gly Ala Asn Gly 65 70 75 80 Glu Cys Gly Arg Gly Glu Pro Gln Cys Ser Pro Glu Gly Pro Ala Arg 85 90 95 Gly Pro Lys Val Ser Phe Ser Cys Arg Gly Ala Ala Ser Gly Pro Ser 100 105 110 Ala Ala Glu Glu Ala Gly Ser Glu Glu Ala Gly Pro Ala Gly Glu Pro 115 120 125 Arg Gly Ser Gln Ala Ser Phe Leu Gln Arg Gln Phe Gly Ala Leu Leu 130 135 140 Gln Pro Gly Val Asn Lys Phe Ser Leu Arg Met Phe Gly Ser Gln Lys 145 150 155 160 Ala Val Glu Arg Glu Gln Glu Arg Val Lys Ser Ala Gly Ala Trp Ile 165 170 175 Ile His Pro Tyr Ser Asp Phe Arg Phe Tyr Trp Asp Phe Thr Met Leu 180 185 190 Leu Phe Met Val Gly Asn Leu Ile Ile Ile Pro Val Gly Ile Thr Phe 195 200 205 Phe Lys Asp Glu Thr Thr Ala Pro Trp Ile Val Phe Asn Val Val Ser 210 215 220 Asp Thr Phe Phe Leu Met Asp Leu Val Leu Asn Phe Arg Thr Gly Ile 225 230 235 240 Val Ile Glu Asp Asn Thr Glu Ile Ile Leu Asp Pro Glu Lys Ile Lys 245 250 255 Lys Lys Tyr Leu Arg Thr Trp Phe Val Val Asp Phe Val Ser Ser Ile 260 265 270 Pro Val Asp Tyr Ile Phe Leu Ile Val Glu Lys Gly Ile Asp Ser Glu 275 280 285 Val Tyr Lys Thr Ala Arg Ala Leu Arg Ile Val Arg Phe Thr Lys Ile 290 295 300 Leu Ser Leu Leu Arg Leu Leu Arg Leu Ser Arg Leu Ile Arg Tyr Ile 305 310 315 320 His Gln Trp Glu Glu Ile Phe His Met Thr Tyr Asp Leu Ala Ser Ala 325 330 335 Val Met Arg Ile Cys Asn Leu Ile Ser Met Met Leu Leu Leu Cys His 340 345 350 Trp Asp Gly Cys Leu Gln Phe Leu Val Pro Met Leu Gln Asp Phe Pro 355 360 365 Ser Asp Cys Trp Val Ser Ile Asn Asn Met Val Asn His Ser Trp Ser 370 375 380 Glu Leu Tyr Ser Phe Ala Leu Phe Lys Ala Met Ser His Met Leu Cys 385 390 395 400 Ile Gly Tyr Gly Arg Gln Ala Pro Glu Ser Met Thr Asp Ile Trp Leu 405 410 415 Thr Met Leu Ser Met Ile Val Gly Ala Thr Cys Tyr Ala Met Phe Ile 420 425 430 Gly His Ala Thr Ala Leu Ile Gln Ser Leu Asp Ser Ser Arg Arg Gln 435 440 445 Tyr Gln Glu Lys Tyr Lys Gln Val Glu Gln Tyr Met Ser Phe His Lys 450 455 460 Leu Pro Ala Asp Phe Arg Gln Lys Ile His Asp Tyr Tyr Glu His Arg 465 470 475 480 Tyr Gln Gly Lys Met Phe Asp Glu Asp Ser Ile Leu Gly Glu Leu Asn 485 490 495 Gly Pro Leu Arg Glu Glu Ile Val Asn Phe Asn Cys Arg Lys Leu Val 500 505 510

Ala Ser Met Pro Leu Phe Ala Asn Ala Asp Pro Asn Phe Val Thr Ala 515 520 525 Met Leu Thr Lys Leu Lys Phe Glu Val Phe Gln Pro Gly Asp Tyr Ile 530 535 540 Ile Arg Glu Gly Thr Ile Gly Lys Lys Met Tyr Phe Ile Gln His Gly 545 550 555 560 Val Val Ser Val Leu Thr Lys Gly Asn Lys Glu Met Lys Leu Ser Asp 565 570 575 Gly Ser Tyr Phe Gly Glu Ile Cys Leu Leu Thr Arg Gly Arg Arg Thr 580 585 590 Ala Ser Val Arg Ala Asp Thr Tyr Cys Arg Leu Tyr Ser Leu Ser Val 595 600 605 Asp Asn Phe Asn Glu Val Leu Glu Glu Tyr Pro Met Met Arg Arg Ala 610 615 620 Phe Glu Thr Val Ala Ile Asp Arg Leu Asp Arg Ile Gly Lys Lys Asn 625 630 635 640 Ser Ile Leu Leu His Lys Val Gln His Asp Leu Ser Ser Gly Val Phe 645 650 655 Asn Asn Gln Glu Asn Ala Ile Ile Gln Glu Ile Val Lys Tyr Asp Arg 660 665 670 Glu Met Val Gln Gln Ala Glu Leu Gly Gln Arg Val Gly Leu Phe Pro 675 680 685 Pro Pro Pro Pro Pro Gln Val Thr Ser Ala Ile Ala Thr Leu Gln Gln 690 695 700 Ala Val Ala Met Ser Phe Cys Pro Gln Val Ala Arg Pro Leu Val Gly 705 710 715 720 Pro Leu Ala Leu Gly Ser Pro Arg Leu Val Arg Arg Ala Pro Pro Gly 725 730 735 Pro Leu Pro Pro Ala Ala Ser Pro Gly Pro Pro Ala Ala Ser Pro Pro 740 745 750 Ala Ala Pro Ser Ser Pro Arg Ala Pro Arg Thr Ser Pro Tyr Gly Val 755 760 765 Pro Gly Ser Pro Ala Thr Arg Val Gly Pro Ala Leu Pro Ala Arg Arg 770 775 780 Leu Ser Arg Ala Ser Arg Pro Leu Ser Ala Ser Gln Pro Ser Leu Pro 785 790 795 800 His Gly Val Pro Ala Pro Ser Pro Ala Ala Ser Ala Arg Pro Ala Ser 805 810 815 Ser Ser Thr Pro Arg Leu Gly Pro Ala Pro Thr Ala Arg Thr Ala Ala 820 825 830 Pro Ser Pro Asp Arg Arg Asp Ser Ala Ser Pro Gly Ala Ala Ser Gly 835 840 845 Leu Asp Pro Leu Asp Ser Ala Arg Ser Arg Leu Ser Ser Asn Leu 850 855 860 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 6 <211> LENGTH: 3102 <212> TYPE: DNA <213> ORGANISM: Murinae gen. sp. <400> SEQUENCE: 6 ccgctccgct ccgcactgcc cggcgccgcc tcgccatgga tgcgcgcggg ggcggcgggc 60 ggccgggcga tagtccgggc acgacccctg cgccggggcc gccgccaccg ccgccgccgc 120 ccgcgccccc tcagcctcag ccaccacccg cgccaccccc gaaccccacg accccctcgc 180 acccggagtc ggcggacgag cccggcccgc gcgcccggct ctgcagccgc gacagcgcct 240 gcacccctgg cgcggccaag ggcggcgcga atggcgagtg cgggcgcggg gagccgcagt 300 gcagccccga gggccccgcg cgcggcccca aggtttcgtt ctcatgccgc ggggcggcct 360 ccgggccctc ggcggccgag gaggcgggca gcgaggaggc gggcccggcg ggtgagccgc 420 gcggcagcca ggctagcttc ctgcagcgcc aattcggggc gcttctgcag cccggcgtca 480 acaagttctc cctgcggatg ttcggcagcc agaaggccgt ggagcgcgag caggaacgcg 540 tgaagtcggc gggggcctgg atcatccacc cctacagcga cttcaggttc tactgggact 600 tcaccatgct gttgttcatg gtgggaaatc tcattatcat tcccgtgggc atcactttct 660 tcaaggacga gaccaccgcg ccctggatcg tcttcaacgt ggtctcggac actttcttcc 720 tcatggactt ggtgttgaac ttccgcaccg gcattgttat tgaggacaac acggagatca 780 tcctggaccc cgagaagata aagaagaagt acttgcgtac gtggttcgtg gtggacttcg 840 tgtcatccat cccggtggac tacatcttcc tcatagtgga gaagggaatc gactccgagg 900 tctacaagac agcgcgtgct ctgcgcatcg tgcgcttcac caagatcctc agtctgctgc 960 ggctgctgcg gctatcacgg ctcatccgat atatccacca gtgggaagag attttccaca 1020 tgacctacga cctggcaagt gcagtgatgc gcatctgtaa cctgatcagc atgatgctac 1080 tgctctgcca ctgggacggt tgcctgcagt tcctggtgcc catgctgcaa gacttcccca 1140 gcgactgctg ggtgtccatc aacaacatgg tgaaccactc gtggagcgag ctctactcgt 1200 tcgcgctctt caaggccatg agccacatgc tgtgcatcgg ctacgggcgg caggcgcccg 1260 agagcatgac agacatctgg ctgaccatgc tcagcatgat cgtaggcgcc acctgctatg 1320 ccatgttcat tgggcacgcc actgcgctca tccagtccct ggattcgtca cggcgccaat 1380 accaggagaa gtacaagcaa gtagagcaat acatgtcctt ccacaaactg cccgctgact 1440 tccgccagaa gatccacgat tactatgaac accggtacca agggaagatg tttgatgagg 1500 acagcatcct tggggaactc aacgggccac tgcgtgagga gattgtgaac ttcaactgcc 1560 ggaagctggt ggcttccatg ccgctgtttg ccaatgcaga ccccaacttc gtcacagcca 1620 tgctgacaaa gctcaaattt gaggtcttcc agcctggaga ttacatcatc cgagagggga 1680 ccatcgggaa gaagatgtac ttcatccagc atggggtggt gagcgtgctc accaagggca 1740 acaaggagat gaagctgtcg gatggctcct atttcgggga gatctgcttg ctcacgaggg 1800 gccggcgtac ggccagcgtg cgagctgaca cctactgtcg cctctactca ctgagtgtgg 1860 acaatttcaa cgaggtgctg gaggaatacc ccatgatgcg gcgtgccttt gagactgtgg 1920 ctattgaccg gctagatcgc ataggcaaga agaactccat cttgctgcac aaggttcagc 1980 atgatctcag ctcaggtgtg ttcaacaacc aggagaatgc catcatccag gagattgtca 2040 aatatgaccg tgagatggtg cagcaggcag agcttggcca gcgtgtgggg ctcttcccac 2100 caccgccacc accgcaggtc acatcggcca ttgccaccct acagcaggct gtggccatga 2160 gcttctgccc gcaggtggcc cgcccgctcg tggggcccct ggcgctaggc tccccacgcc 2220 tagtgcgccg cgcgccccca gggcctctgc ctcctgcagc ctcgccaggg ccacccgcag 2280 caagcccccc ggctgcaccc tcgagccctc gggcaccgcg gacctcaccc tacggtgtgc 2340 ctggctctcc ggcaacgcgc gtggggcccg cattgcccgc acgtcgcctg agccgcgcct 2400 cgcgcccact gtccgcctcg cagccctcgc tgccccatgg cgtgcccgcg cccagccccg 2460 cggcctctgc gcgcccggcc agcagctcca cgccgcgcct gggacccgca cccaccgccc 2520 ggaccgccgc gcccagtccg gaccgcaggg actcagcctc gccgggcgct gccagtggcc 2580 tcgacccact ggactctgcg cgctcgcgcc tctcttccaa cttgtgaccc ttgagcgccg 2640 ccccgcgggc cgggcggggc cgtcatccac accaaagcca tgcctcgcgc cgcccgcccg 2700 tgcccgtgca gaagccatag agggacgtag gtagcttagg aggcgggcgg ccctgcgccc 2760 ggctgtcccc ccatcgccct gcgcccaccc ccatcgcccc tgccccagcg gcggccgcac 2820 gggagaggga ggggtgcgat cacctcggtg cctcagcccc aacctgggac agggacaggg 2880 cggccctggc cgaggacctg gctgtgcccc gcatgtgcgg tggcctccga ggaagaatat 2940 ggatcaagtg caatacacgg ccaagccggc gtgggggtga ggctgggtcc ccggccgtcg 3000 ccatgaatgt actgacgagc cgaggcagca gtggccccca cgccccatta acccacaacc 3060 ccattccgcg caataaacga cagcattggc aaaaaaaaaa aa 3102 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 7 <211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(17) <223> OTHER INFORMATION: Description of Artificial SequencePrimer <400> SEQUENCE: 7 gccaatacca ggagaag 17 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 8 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(19) <223> OTHER INFORMATION: Description of Artificial SequencePrimer <400> SEQUENCE: 8 tgagtagagg cgacagtag 19 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 9 <211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(24) <223> OTHER INFORMATION: Description of Artificial SequencePrimer <400> SEQUENCE: 9 agtggcctcg acccactgga ctct 24 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 10 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(25) <223> OTHER INFORMATION: Description of Artificial SequencePrimer <400> SEQUENCE: 10 ccgcctccta agctacctac gtccc 25

Claims

What is claimed is:

1. A process comprising a) providing, in a suitable container, cells that express a hyperpolarization-activated cation channel; b) hyperpolarizing the cells in the presence of a potential-sensitive fluorescent dye and an isoosmolar sodium-ion-free buffer; c) optionally, determining the membrane potential of the cells; d) simultaneously adding sodium ions and a sample containing at least one substance to be tested for its ability to modulate the activity of the cation channel; e) determining the membrane potential of the cells; f) determining whether the membrane potential changed upon simultaneous addition of sodium ions and the substance(s); and g) optionally, recording the change in membrane potential, wherein a change in membrane potential indicates the presence of at least one substance in the sample that modulates the activity of the cation channel.

2. The process of claim 1, wherein step c) is performed.

3. The process as claimed in claim 1, wherein the isoosmolar sodium-ion-free buffer comprises a potassium salt.

4. The process as claimed in claim 1, wherein the isoosmolar sodium-ion-free buffer comprises potassium ions at a concentration of at least 0.8 mM.

5. The process as claimed in claim 1, wherein the isoosmolar sodium-ion-free buffer comprises potassium ions at a concentration of at least 5 mM.

6. The process as claimed in claim 1, wherein the isoosmolar sodium-ion-free buffer comprises choline chloride or NMDG (N-methyl-D-glucamine).

7. The process as claimed in claim 1, wherein the potential-sensitive dye is an oxonol derivative.

8. The process as claimed in claim 7, wherein the oxonol derivative is a 3-bis-barbituric acid oxonol.

9. The process as claimed in claim 8, wherein the 3-bis-barbituric acid oxonol is bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBac.sub.4 (3)], bis-(1,3-diethylthiobarbituric acid)trimethine oxonol, bis-(1,3-dibutylbarbituric acid)pentamethine oxonol, or a combination of these.

10. The process as claimed in claim 1, wherein the potential-sensitive fluorescent dye used is suitable for use in fluorescent imaging plate reader system.

11. The process as claimed in claim 1, wherein cells having an elevated intracellular cAMP concentration are used.

12. The process as claimed in claim 11, wherein the intracellular cAMP concentration is increased by addition of dibutyryl-cAMP or 8-bromo-cAMP.

13. The process as claimed in claim 11, wherein the intracellular cAMP concentration is increased by addition of an adenylate cyclase activator.

14. The process as claimed in claim 11, wherein the intracellular cAMP concentration is increased by addition of forskolin.

15. The process as claimed in claim 14, wherein the intracellular cAMP concentration is increased by addition of from 1 pM to 100 pM of forskolin.

16. The process as claimed in claim 11, wherein the intracellular cAMP concentration is increased by addition of receptor ligands.

17. The process as claimed in claim 1, wherein the hyperpolarization-activated cation channel is HCN1, HCN2, HCN3, HCN4, KAT1, or a heteromultimer of these channels.

18. The process as claimed in claim 1, wherein the hyperpolarization-activated cation channel is a human hyperpolarization-activated cation channel.

19. The process as claimed in claim 1, wherein the cells are mammalian cells.

20. The process as claimed in claim 19, wherein the cells are CHO or HEK cells.

21. The process as claimed in claim 1, wherein the cells contain a plasmid which comprises the cDNA of a hyperpolarization-activated cation channel.

22. The process as claimed in claim 1, wherein the cells comprise a second plasmid, which comprises the cDNA of the same hyperpolarization-activated cation channel.

23. The process as claimed in claim 22, wherein the cells comprise a second plasmid, which comprises the cDNA of a different hyperpolarization-activated cation channel, such that heteromultimeric HCN channels can be formed.

24. The process as claimed in claim 1, wherein the cells comprise a plasmid, which comprises synthetic cDNA encoding at least part of at least two different cation channels.

25. The process as claimed in claim 1, wherein a change in membrane potential is measured using a potential-sensitive fluorescent dye.

26. The process as claimed in claim 25, wherein the potential-sensitive fluorescent dye is an oxonol derivative.

27. The process as claimed in claim 26, wherein the oxonol derivative is 3-bis-barbituric acid oxonol.

28. The process as claimed in claim 1, wherein at least one measurement is carried out in a Fluorescent Imaging Plate Reader (FLIPR).

29. The process as claimed in claim 1, wherein the change of the membrane potential of at least two cells is compared.

30. The process as claimed in claim 1, wherein the process is a high-throughput screening process.

31. The process as claimed in claim 1, wherein the hyperpolarization-activated cation channel is HCN1, HCN2, HCN3, HCN4, KAT1, or a heteromultimer of these channels.